Branched linker for protein drug conjugates

ABSTRACT

The present invention relates to method for connecting a protein and a drug to a protein drug conjugate, wherein the drug is linked to the protein through a specific branched linker, said branched linker comprises a peptide chain and is derived from o-hydroxy p-amino benzylic alcohol, wherein the peptide chain is connected to the phenyl ring via the p-amino group, the drug is connected to the phenyl ring via the benzylic alcohol moiety, and the protein is connected to the phenyl ring via the o-hydroxy group; further to a process for the preparation of said protein-drug-conjugates via various intermediates, to the pharmaceutical use of such protein drug conjugates, such as methods of controlling the growth of undesirable cells, to pharmaceutical compositions comprising such protein drug conjugates, and to intermediates of the preparation of the protein drug conjugates.

RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 14/001,237 having a filing date of Aug. 23, 2013, which is anational stage entry of International Patent Application No.PCT/EP2012/053039 having a filing date of Feb. 23, 2012, which claimsfiling benefit of International Patent Application PCT/CN2011/071287having a filing date of Feb. 25, 2011; PCT/CN2011/077863 having a filingdate of Aug. 1, 2011; PCT/CN2011/081857 having a filing date of Nov. 7,2011, which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 23, 2014, isnamed LZA-47-PCT-US_SL.txt and is 862 bytes in size.

The present invention relates to method for connecting a protein and adrug to a protein drug conjugate, wherein the drug is linked to theprotein through a specific branched linker, said branched linkercomprises a peptide chain and is derived from o-hydroxy p-amino benzylicalcohol, wherein the peptide chain is connected to the phenyl ring viathe p-amino group, the drug is connected to the phenyl ring via thebenzylic alcohol moiety, and the protein is connected to the phenyl ringvia the o-hydroxy group;

further to a process for the preparation of said protein-drug-conjugatesvia various intermediates, to the pharmaceutical use of such proteindrug conjugates, such as methods of controlling the growth ofundesirable cells, to pharmaceutical compositions comprising suchprotein drug conjugates, and to intermediates of the preparation of theprotein drug conjugates.

Most drugs used for chemotherapy have severe side-effects which limittheir efficacy and use. Linking such payloads, i.e. pharmaceuticallyactive compounds, such as drugs, to targeting agents, in particularmonoclonal antibodies, affords novel antibody drug conjugates (ADC) fore.g. cancer therapy. Tissue-specificity is typically governed by themonoclonal antibody (mAb) component, while the drug provides thetherapeutic effect. The efficiency and tolerability of ADCs is dependenton the interplay between the target antigen, drug potency andconjugation technology. In particular, linker chemistry stronglyinfluences the ADC specificity and safety.

Instead of chemically labile linkers, which have limited stability inphysiological extracellular conditions, such as hydrazone- anddisulfide-based linkers, linkers, which are stable in physiologicalextracellular conditions, especially which have high plasma stability,are desired for improving the therapeutic applicability, because thedrug should be released only within the cell, which is targeted by theprotein, to which the drug is linked, and not outside of the cell.

Non cleavable linkers have disadvantages: ADC internalization followedby complete hydrolysis of the polypeptide backbone of the mAb isrequired for payload release, and reduced efficacy may be encounteredwhen ADC internalization is poor. Thus, ADCs bearing non-cleavablelinkers are highly dependent on the biology of the target cell. Also,not all payloads retain their biological activity when attached to thelast amino acid of the mAb as is the case after mAb degradation.

In order to allow the ADC to deliver the payload not only to tumor cellsbut also to adjacent antigen-negative cells, i.e. bystander effect, thereleased payload must readily diffuse through hydrophobic cellmembranes, which is not the case, when ADCs with non-cleavable linkersrelease their payloads in form of an amino acid bearing drug with theamino acid being in a zwitterionic state, i.e. having a positivelycharged ammonium and a negatively charged carboxylate.

Therefore it is desired to have a linker which shows high plasmastability, and which releases the drug without the drug being chemicallymodified after its release.

Furthermore it is desired to have protein drug conjugates, which showreduced agglomeration or aggregation, which would impair theirperformance.

EP 624377 A discloses a drug ligand conjugate, wherein the linkercomprises a linear peptide.

Fanny Guzman et al, Electronic Journal of Biotechnology, 2007, 10,279-314; Yoshio Okada, Current Organic Chemistry, 2001, 5, 1-43; U.S.Pat. No. 6,897,289 B and the text books “Houben-Weyl Synthesis ofPeptides and Peptidomimetics (Methods in Organic Chemistry)”, MurrayGoodman et al., Thieme Publishing Group, 2001, in particular VolumesE22a and E22b; disclose protecting groups, peptide bond formation, thesynthesis of peptides and proteins detailing general and specificmethods, and analytical techniques used to determine the structure andcomposition of peptides.

Known linkers comprising linear peptide chains still show deficiencies.There was a need for linkers and for protein drug conjugates based onsuch linkers, which show improved performance. Surprisingly, specificbranched linkers derived from o-hydroxy p-amino benzylic alcohol showthe desired performance.

In the following text, the following abbreviations are used, if nototherwise stated:

DCC N,N′-dicyclohexylcarbodiimide

EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

Fmoc 9-fluorenylmethoxycarbonyl

Boc tert-butoxycarbonyl

Boc2O di-tert-butyl dicarbonate

Cit citrulline

NBS N-bromosuccinimide

NHS compound of formula (HOSu), N-hydroxysuccinimide

NIS N-iodosuccinimide

—OTs tosylate

—OMs mesylate

—OTf triflate

PBS phosphate buffered saline

Red-Al sodium bis(2-methoxyethoxy)aluminium hydride

TCEP tris(2-carboxyethyl)phosphine hydrochloride

Tos or Ts Tosyl or p-toluenesulfonyl

TsCl Tosyl chloride or p-toluenesulfonyl chloride

Z or Cbz benzyloxycarbonyl

Subject of the invention is a method (MI) for connecting a ligand LIwith a drug DR,

-   LI is selected from the group consisting of amino acids LI-AA, mono-    or polyclonal antibodies LI-Ab, antibody fragments LI-AbFrag,    proteins LI-Prot and peptides LI-Pep;-   DR is a pharmaceutically active drug;-   characterized that a linker LIN is used to covalently connect LI    with DR;-   LIN comprises a connecting group CG2;-   CG2 is derived from o-hydroxy p-amino benzylic alcohol and is a    connecting group of formula (CG2-1);

-   (***) denotes the connecting site which is used to connect LI;-   (****) denotes the connecting site which is used to connect DR;-   (******) denotes the connecting site to which a linear peptide is    connected, said peptide has 2 to 8 amino acid residues;-   (4) denotes the p-amino group of the o-hydroxy p-amino benzylic    alcohol from which CG2 is derived.-   Further subject of the invention is a method (MI), with the    method (MI) as defined herein, also with all its preferred    embodiments,-   wherein LI is covalently connected with DR in form of a compound of    formula (I);

-   CG2 is as defined herein, also with all its preferred embodiments;-   CG1 is a connecting group selected from the group consisting of    connecting group of formula (CG1-I), connecting group of formula    (CG1-II), connecting group of formula (CG1-III) and connecting group    of formula (CG1-IV);

-   m30 and m32 are identical or different and independently from each    other 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;-   (*) in the formulae of CG1 denotes the bond between T1 and CG1,-   the covalently connected LI forms in compound of formula (I) a    ligand residue LIRes, which is covalently connected to CG1 via T1;-   LI is as defined herein, also with all its preferred embodiments,    and is a compound of formula (LIRes-T1-H);    LIRes-T1-H  (LIRes-H)-   LIRes is selected from the group consisting of amino acid residue    LIRes-AA, mono- or polyclonal antibody residue LIRes-Ab, antibody    fragment residue LIRes-AbFrag, protein residue LIRes-Prot and    peptide residue LIRes-Pep;-   LI has a functional group selected from the group consisting of SH,    OH or NH₂, which forms in formula (I) the T1, the T1 is bonded to    CG1 via the bond (*);-   T1 is —S—, —O— or —NH—;-   n1 is 0 or 1;-   SG is a spacer group selected from the group consisting of spacer    group of formula (SG-II) and spacer group of formula (SG-III);

-   m1 and m2 are identical or different and independently from each    other 0 or 1;-   m10, m11 and m12 are identical or different and independently from    each other 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;-   with the proviso, that m2 and m10 are not simultaneously 0;-   with the proviso, that m1, m11 and m12 are not simultaneously 0;-   SGPEG is a connecting group of formula (SGPEG-I);    CH₂—CH₂—O    _(m20)CH₂—CH₂—  (SGPEG-I)-   m20 is 1, 2, 3, 4, 5 or 6;-   n2 is 0 or 1;-   T4 is —O—;-   CG3 is selected from the group consisting of connecting group of    formula (CG3-I) and connecting group of formula (CG3-II);

-   R5 and R6 are identical or different and independently from each    other C₁₋₄ alkyl;-   for n1 being 1, the (**) in the formulae of CG1 and in the formulae    of SG denote the bond between to CG1 and SG, and the (***) in the    formula of SG and in the formula of CG2 denote the bond between SG    and CG2; in case that CG1 is a connecting group of formula (CG1-I),    the nitrogen atom denoted with (**) in SG forms an endocyclic    nitrogen atom, thereby replacing the hydrogen atom of said nitrogen    atom by an endocyclic bond;-   for n1 being 0, the (**) in the formulae of CG1 and the (***) in the    formula of CG2 denote the bond between CG1 and CG2;-   with the proviso, that in case that n1 is 0, then CG1 is not a    connecting group of formula (CG1-I);-   for n2 being 1, the (****) in the formula of CG2 and in the formula    of CG3 denote the bonds, with which CG2 and CG3 are bonded to T4;    and the (*****) in the formula of CG3 denotes the bond between CG3    and T2;-   for n2 being 0, the (****) in the formula of CG2 denotes the bond    between CG2 and T2;-   the (******) in the formula of CG2 denotes the bond between CG2 and    AA^(n4);-   the covalently connected DR forms in compound of formula (I) a drug    residue DRRes, which is covalently connected to CG2 via T2;-   DR is as defined herein, also with all its preferred embodiments,    and is a compound of formula (DRRes-T2-H);    H-T2DRRes  (DRRes-T2-H)-   DRRes is a drug residue derived from DR;-   DR has a functional group selected from the group consisting of    —N(R4)H, —OH or —SH, which forms in formula (I) the T2;-   T2 is —N(R4)-, —O— or —S—;-   R4 is H or C₁₋₄ alkyl;-   n3 is 2, 3, 4, 5, 6, 7 or 8;-   n4 is an integer from 1 to n3;-   AA^(n4) is an amino acid residue, with n4 being the index of said    amino acid residue, and (AA^(n4))_(n3) is a linear peptide with n3    amino acid residues AA^(n4) and with n4 denoting the position of the    amino acid residue AA^(n4) in the peptide starting from CG2, in    which peptide the individual amino acid residues are connected to    each other via a peptide bond, with AA¹ being the first amino acid    residue in the chain and being connected to CG2 via the bond    (******), with the bond (******) being an amid bond of the    carboxylic acid group of AA¹ with the amino group denoted with (4)    of CG2, and with AA^(n3) being the last amino acid residue in the    chain, and with the individual AA^(n4) being independently from each    other identical or different;-   (3) denotes the N-terminal amino group of AA^(n3);-   R1 and R2 are identical or different and independently from each    other selected from the group consisting of hydrogen, C₁₋₄ alkyl,    C(O)—(CH₂—O—)_(m5)-(GRPEG)_(m4)-R3 and PGN;-   R3 is C₁₋₄ alkyl;-   m4 is 0 or 1;-   m5 is 0 or 1;-   PGN is a protecting group;-   GRPEG is a connecting group of formula (GRPEG-I);    CH₂—CH₂—O    _(m21)  (GRPEG-I)-   m21 is 1, 2, 3, 4, 5 or 6;-   LI, DR and CG2 are as defined herein, also with all their preferred    embodiments.

Preferably, LIN is compound of formula (LIN).

-   Preferably, the drug DR is selected from group consisting of    cytotoxic agents, other chemotherapeutic and antimetastatic agents.

Preferably, the other chemotherapeutic and antimetastatic agents areselected from the group consisting of tyrosine kinase inhibitors andRac1 inhibitors.

Preferably, tyrosine kinase inhibitors are selected from the group ofactive pharmaceutical ingredients (API) consisting of Imatinib,Lapatimib, Sunitimib, Nilotimib and Dasatimib.

Preferably, Rac1 inhibitors is NSC 23766.

Preferable cytotoxic agents are those used for cancer therapy.

-   Preferable classes of cytotoxic agents include, for example, the    enzyme inhibitors such as the anthracycline family of drugs, the    bleomycins, the cytotoxic nucleosides, dihydrofolate reductase    inhibitors, differentiation inducers, DNA cleavers, DNA    intercalators, diynenes, the mitomycins, the podophyllotoxins, the    pteridine family of drugs, taxols, thymidylate synthase inhibitors,    topoisomerase inhibitors, and the vinca drugs.-   Preferable useful members of various classes of cytotoxic agents are    selected from the group consisting of N8-acetyl spermidine,    actinomycin, 9-amino camptothecin, aminopterin, anguidine,    anthracycline, auristatin, bleomycin, calicheamycin, camptothecin    (lactone or ring-opened form of the lactone), carminomycin, CC-1065,    clofaribine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazide,    cyclopropabenzindol-4-one (CBI), cytarabine, cytosine arabinoside,    daunorubicin, dichloromethotrexate, n-(5,5-diacetoxy-pentyl)    doxorubicin,    -   1,8-dihydroxy-bicyclo[7.3.1] trideca-4-9-diene-2,6-diyne-13-one,        difluoronucleosides, doxorubicin, duocarmycin, epirubicin,        esperamicin, etoposide, 5-fluorouracil, irinotecan,        leurosideine, leurosine, maytansine, melphalan,        6-mercaptopurine, methopterin, methotrexate, mitomycin A,        mitomycin C, morpholine-doxorubicin, nemorubicin,        podophyllotoxin and podophyllotoxin derivatives such as        etoposide or etoposide phosphate, retinoic acid, saporin,        tallysomycin, vinblastine, vincristine, vindesine, taxane, such        as taxol or paclitaxel, taxotere or docetaxel, and taxotere        retinoic acid, and analogues and derivatives thereof.-   More preferable cytotoxic agents are selected from the group    consisting of anthracycline, auristatin, calicheamycin,    cyclopropabenzindol-4-one (CBI), doxorubicin, duocarmycin,    maytansine, mitomycin C, taxol, and analogues and derivatives of    these substances.

Preferably, R4 is —H.

Preferably, T1 is —NH— or —S—.

-   Preferably, n2 is 1 and T2 is —NH—, —O— or —S— and is connected via    the bond (****) with CG3; more preferably, T2 is —NH— or —S—.

Doxorubicin has the CAS number 23214-92-8 and is the compound of formula(DOXO).

Compound of formula (DOXO) is also used in form of its hydrochloridesalt.

-   Doxorubicin can be connected to CG3 or CG2 respectively via one of    its functional groups, e.g. via one of the functional groups denoted    with (d1), (d2), (d3) and (d4) in formula (DOXO).-   The functional groups denoted with (d1), (d2), (d3) and (d4)    function then as the T2 in formula (I).-   Preferably doxorubicin is connected via the amino group denoted with    (d1) in formula (DOXO) with CG3 via the bond (*****).

Maytansine has the CAS number 35846-53-8 and the formula (MAYT).

-   Maytansine can be connected to CG3 or CG2 respectively via the —OH    denoted with (m2) in formula (MAYT).-   Or the —C(O)—CH₃ group denoted with (m1) in the formula (MAYT) is    exchanged against an acyl group, said acyl group has a nucleophilic    group —SH, —NH₂ or —OH, which again is the connected to CG3 or CG2    respectively.-   The —OH denoted with (m2) or said nucleophilic group of the acyl    group function then as the T2 in formula (I).

A preferred taxane is taxol with the CAS number 33069-62-4 and theformula (TAXO).

-   Taxol can be connected to CG3 or CG2 respectively via one of the —OH    denoted with (t1), (t2) and (t3) in formula (TAXO). Said —OH    functions then as the T2 in formula (I).-   In one preferred embodiment, n2 is 0, T2 is —O— and is connected via    the bond (****) with CG2, and DRRes is derived from DR, with DR    being compound of formula (TAXO).-   In another preferred embodiment, n2 is 1, T2 is —O— and is connected    via the bond (*****) with CG3, CG3 is the connecting group of    formula (CG3-II) and DRRes is derived from DR, with DR being    compound of formula (TAXO), one possible Taxol intermediate for this    embodiment is the compound of formula (TAXO-t1-1).

-   A Camptothecin is (S)-(+)-camptothecin, which has the CAS number    7689-03-4 and is the compound of formula (CAMPTO).

-   Camptothecin can be connected to CG3 or the CG2 respectively via the    functional group denoted with (c1) in formula (CAMPTO).

The functional group denoted with (c1) functions then as the T2 informula (I).

-   In one preferred embodiment, n2 is 0, T2 is —O— and is connected via    the bond (****) with CG2, and DRRes is derived from DR, with DR    being compound of formula (CAMPTO).-   In another preferred embodiment, n2 is 1, T2 is —O— and is connected    via the bond (*****) with CG3, CG3 is the connecting group of    formula (CG3-II) and DRRes is derived from DR, with DR being    compound of formula (CAMPTO).-   In one preferred embodiment, CG1 is the connecting group of formula    (CG1-I), (CG1-III) or (CG1-IV) and the sulphur atom of the side    chain of a Cys residue of LIRes is T1 and is connected via the bond    (*) to CG1.-   In another preferred embodiment, CG is the connecting group of    formula (CG1-II), and T1 is —N— or —O— of LIRes and is connected to    CG1 via the bond (*). This amino or hydroxyl group of LIRes    connected to CG1 is preferably the N-terminal amino group of LIRes    or an amino or hydroxy group of a side chain of an amino acid    residue of LIRes. Preferably, in case T1 is an amino group of a side    chain of an amino acid residue of LIRes connected to CG1, said amino    acid residue of LIRes is preferably a Lys; in case T1 is an hydroxyl    group of a side chain of an amino acid residue of LIRes connected to    CG1, said amino acid residue of LIRes is preferably a Tyr, Ser or    Thr.-   Preferably, LI is selected from the group consisting of mono- or    polyclonal antibodies LI-Ab, antibody fragments LI-AbFrag, proteins    LI-Prot and peptides LI-Pep; and-   LIRes is selected from the group consisting of mono- or polyclonal    antibody residue LIRes-Ab, antibody fragment residue LIRes-AbFrag,    protein residue LIRes-Prot and peptide residue LIRes-Pep.

In the case that LIRes is LIRes-AA, LIRes is preferably an alpha aminoacid residue.

LIRes can be preferably connected to CG1 via one of two possiblefunctional groups of LIRes: via a N-terminal amino group or via afunctional group of a side chain of LIRes, if LIRes has such a sidechain with a functional group. This functional group, which connectsLIRes with CG1, is the T1. In case that LIRes is connected via afunctional group of a side chain of LIRes, said side chain is preferablya side chain of a Cys, Lys, Tyr, Ser or Thr residue of LIRes.

In case of LIRes being LIRes-AA, the remaining functional groups ofLIAARes-AA, which are not connected to CG1, can be protected by aprotecting group commonly used in peptide chemistry, e.g. anon-connected amino group can carry a acetate, a non-connected carboxygroup can be esterified with a C₁₋₄ alcohol, a non-connected functionalgroup of a side chain can carry a side chain protecting groupconventionally used in peptide chemistry.

More preferably, LIRes-AA is an alpha amino acid residue with a sidechain having a functional group and is connected via this functionalgroup with CG1, even more preferably LIRes-AA is derived from Cys, Lys,Tyr, Ser or Thr.

-   Preferably, in the case, that LIRes is connected via the side chain    of a Cys residue of LIRes, T1 is formed by the sulfur atom of the    side chain of said Cys residue and is connected through the bond (*)    with CG1, preferably with CG1 being the connecting group of formula    (CG1-I), (CG1-III) or (CG1-IV); or-   in the case, that LIRes is connected via the side chain of a Lys,    Tyr, Ser or Thr residue of LIRes, T1 is formed by the nitrogen or    oxygen atom of the side chain of said Lys, Tyr, Ser or Thr residue    and is connected through the bond (*) with CG1, preferably with CG1    being the connecting group of formula (CG1-II).

LIRes-Pep can be derived from a cell-penetrating peptide.

LIRes-AB and LIRes-ABFrag are preferably derived from antibodies andantibody fragments used in treatment of diseases, preferably in cancertreatment.

-   Preferably, m30 and m32 are identical or different and independently    from each other 2, 3, 4, 5 or 6.

Preferably, m30 is 2 when CG1 is a connecting group of formula (CG1-IV).

Preferably, m32 is 2 when CG1 is a connecting group of formula (CG1-II).

Preferably, n1 is 1.

Preferably, m1 and m2 are 0 or 1.

-   Preferably, m10, m11 and m12 are identical or different and    independently from each other 0, 1, 2, 3, 4, 5 or 6.

Preferably, m20 is 1, 2, 3 or 4.

More preferably,

m10 is 6 and m2 is 0; or

m2 is 1, m10 is 0 and m20 is 1; or

m1 is, m11 is 1, m12 is 1 and m20 is 3; or

m1 is 0, m11 is 3, m12 is 0.

Preferably, R5 and R6 are CH₃.

-   In a particular embodiment, CG1 is a connecting group of formula    (CG1-I), SG is a spacer group of formula (SG-II), m10 is 6 and m2 is    0.-   In another particular embodiment, CG1 is a connecting group of    formula (CG1-II) with m32 being 2 or a connecting group of formula    (CG1-III), or a connecting group of formula (CG1-IV) with m30 being    2, and SG is a spacer group of formula (SG-II), m10 is 0, m2 is 1    and m20 is 1.-   In another particular embodiment, CG1 is a connecting group of    formula (CG1-II) with m32 being 2 or a connecting group of formula    (CG1-III), or a connecting group of formula (CG1-IV) with m30 being    2, and SG is a spacer group of formula (SG-III) with m1 being 1, m11    being 1, m12 being 1 and m20 being 3, or SG is a spacer group of    formula (SG-III) with m1 being 0, m11 being 3 and m12 being 0.

Preferably, n3 is 2, 3, 4, 5 or 6; more preferably 2, 3 or 4; even morepreferably, n3 is 2 or 3.

Preferably, AA^(n4) is an alpha amino acid residue.

-   More preferably, AA^(n4) is selected from the group consisting of    alanine, valine, leucine, isoleucine, methionine, phenylalanine,    tryptophan, proline, lysine, lysine side chain protected with acetyl    or formyl, arginine, arginine side chain protected, preferably    protected with tosyl or nitro groups, histidine, ornithine,    ornithine side chain protected, preferably protected with acetyl or    formyl, and citrulline.-   Even more preferably, AA^(n4) is alanine, glycine, phenylalanine,    valine, lysine, leucine, tryptophan, arginine, side-chain protected    arginine or citrulline, especially alanine, glycine, phenylalanine,    valine, lysine or citrulline.

In case that AA^(n4) has a side chain with a functional group, thisfunctional group can be protected by a protecting group commonly usedfor protecting functional groups of side chains of amino acids.

In case of Lys, the side chain is preferably protected with acetyl orformyl

Examples of peptides for the (AA^(n4))_(n3) peptide chain are Phe-Lys,Val-Lys, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit,Phe-Cit, Leu-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly (SEQ ID NO: 1),Ala-Leu-Ala-Leu (SEQ ID NO: 2), Phe-N9-tosyl-Arg and Phe-N9-Nitro-Arg,preferably Phe-Lys, Val-Lys, Val-Cit and D-Phe-L-Phe-Lys; any Lys sidechain being optionally protected, preferably with acetyl.

Especially preferably, n3 is 2 or 3, and AA^(n4) is alanine, glycine,valine or citrulline;

more especially,

n3 is 2 and AA¹ is citrulline and AA² is valine or alanine; or

n3 is 3 and AA¹ is citrulline, AA² is valine and AA³ is glycine.

Preferably, m4 is 1.

Preferably, R3 is methyl.

-   Preferably, R1 and R2 are identical or different and independently    from each other selected from the group consisting of hydrogen,    methyl, C(O)—(CH₂—O—)_(m5)-(GRPEG)_(m4)-CH₃ and PGN.-   Preferably, m21 is 2, 3 or 4.-   Preferably, PGN is a protecting group commonly used in peptide    chemistry for protecting the N-terminus of a peptide or for    protecting the alpha amino group of an alpha amino acid used as    building block in peptide synthesis.

More preferably, PGN is selected from group consisting of Boc, Fmoc andZ.

-   Even more preferably, R1 is hydrogen, methyl, acetyl or    C(O)—(CH₂—O—)_(m5)-(GRPEG)_(m4)-CH₃ with m4 being 1 and m21 being 3,    and R2 is hydrogen or methyl.

Especially,

-   -   R1 is acetyl and R2 is hydrogen; or    -   R1 and R2 are methyl; or    -   R1 is C(O)—(CH₂—O—)_(m5)-(GRPEG)_(m4)-CH₃ with m5 being 0, m4        being 1 and m21 being 3, and R2 is hydrogen; or    -   R1 is C(O)—(CH₂—O—)_(m5)-(GRPEG)_(m4)-CH₃ with m5 being 1, m4        being 1 and m21 being 2, and R2 is hydrogen.

Further subject of the invention is a method (MI);

-   wherein compound of formula (I) is prepared in a step (MI);-   step (MI) comprises a reaction (MI), wherein a compound of    formula (II) is reacted with a compound of formula (LIRes-T1-H);

-   CG1M is a connecting group selected from the group consisting of    connecting group of formula (CG1M-I), connecting group of formula    (CG1M-II), connecting group of formula (CG1M-III) and connecting    group of formula (CG1M-IV);

-   X1 is Cl, Br or I;-   SG, n1, n4, n3, AA^(n4), (3), R1, R2, T4, CG3, n2, T2, DRRes, m30    and m32 are as defined herein, also with all their preferred    embodiments;-   CG2 is as defined herein, also with all its preferred embodiments.

Preferably, X1 is Cl or Br, more preferably X1 is Br.

Preferably, CG1M is a connecting group of formula (CG1M-IV).

More preferably, CG M is a connecting group of formula (CG1M-IV) and m30is 2.

-   In a particular embodiment, CG1M is a connecting group of formula    (CG1M-I), SG is a spacer group of formula (SG-II), m10 is 6 and m2    is 0.-   In another particular embodiment, CG1M is a connecting group of    formula (CG1M-II) with m32 being 2 or a connecting group of formula    (CG1M-III) or a connecting group of formula (CG1M-IV) with m30 being    2, and SG is a spacer group of formula (SG-II), m10 is 0, m2 is 1    and m20 is 1.-   In another particular embodiment, CG1M is a connecting group of    formula (CG1-II) with m32 being 2 or a connecting group of formula    (CG1-III), or a connecting group of formula (CG1M-IV) with m30 being    2, and SG is a spacer group of formula (SG-III) with m1 being 1, m11    being 1, m12 being 1 and m20 being 3, or SG is a spacer group of    formula (SG-III) with m1 being 0, m11 being 3 and m12 being 0.

Preferably, the reaction temperature of reaction (MI) is from 0 to 150°C., more preferably from 5 to 50° C., even more preferably from 10 to40° C.

Preferably, the reaction time of reaction (MI) is from 1 min to 168 h,more preferably from 10 min to 24 h, even more preferably from 15 min to3 h.

Reaction (MI) is usually done in a solvent (MI).

Preferably, solvent (MI) is selected from the group consisting of water,N,N-dimethylacetamide, N,N-dimethylformamide, N,N-dimethylsulfoxide andmixtures thereof. In case of water, the water can comprise a buffer(MI), preferably the buffer (MI) is a buffer conventionally used inprotein chemistry, more preferably buffer (MI) is derived from abuffering substance selected from the group consisting of acetic acid,citric acid, dithiothreitol (DTT), ethylenediaminetetraacetic acid(EDTA), glycine, histidine, phosphoric acid (incl. phosphate bufferedsaline, PBS), polysorbate 20, polysorbate 80, saccharose, sodiumchloride, succinic acid, trehalose, tris-(hydroxymethyl)-aminomethane,mixtures thereof and salts thereof.

The salts of said buffering substance are preferably sodium salt,potassium salts or HCl salts. Preferably, the amount of solvent (MI) isfrom 5 to 10000 fold, more preferably from 10 to 5000 fold, even morepreferably from 50 to 500 fold, of the weight of compound of(LIRes-T1-H).

Preferably, in reaction (MI), from 1 to 100 mol equivalents, morepreferably from 2 to 20 mol equivalents, even more preferably from 3 to10 mol equivalents, of compound of formula (II) are used, the molequivalents being based the mol of compound of formula (LIRes-T1-H).

Reaction (MI) can be done in the presence of TCEP.

TCEP is preferably used, when LI is a mono- or polyclonal antibodiesLI-Ab or an antibody fragments LI-AbFrag.

Preferably, in reaction (MI), from 0.5 to 20 mol equivalents, morepreferably from 1 to 10 mol equivalents, even more preferably from 1.5to 5 mol equivalents, of TCEP are used, the mol equivalents being basedthe mol of compound of formula (LIRes-T1-H)

After the reaction (MI), compound of formula (I) can be isolated bystandard methods such as washing, extraction, filtration, concentrationand drying. The compound of formula (I) can be purified before or afterisolation, preferably by chromatography or crystallization from anappropriate solvent.

Alternatively, compound of formula (I) can be purified by standardmethods such as filtration, ultrafiltration, diafiltration andchromatography, and ca be stored or further used as a solution.

-   Further subject of the invention is a method (MII) for the    preparation of compound of formula (II), with the compound of    formula (II) as defined herein, also with all its preferred    embodiments,-   wherein-   in case that n2 is 1 and CG3 is a connecting group of formula    (CG3-I), then method (MII) comprises a step (MIIa) and a step    (MIIb);-   in case that n2 is 1 and CG3 is a connecting group of formula    (CG3-II), then method (MII) comprises the step (MIIa), a step    (MIIc), a step (MIId) and a step (MIIe);-   in case that n2 is 0 and CG1M is a connecting group of formula    (CG1M-IV) then method (MII) comprises one step, a step (MII0-IV), or    two steps, a step (MII0-I-IVa) and a step (MII0-I-IVb);-   in case that n2 is 0 and CG1M is a connecting group of formula    (CG1M-III) then method (MII) comprises a step (MII0-III);-   in case that n2 is 0 and CG1M is a connecting group of formula    (CG1M-II) then method (MII) comprises two steps, a step (MII0-IIa)    and a step (MII0-IIb), or one step, a step (MII0-IIc);-   in case that n2 is 0 and CG1M is a connecting group of formula    (CG1M-I) then method (MII) comprises one step, a step (MII0-I), or    two steps, a step (MII0-I-IVa) and a step (MII0-I-IVb);-   step (MIIa) comprises a reaction (MIIa), wherein a compound of    formula (III) is reacted with a compound (II-I);

-   n2 in formula (III) is as defined herein, also with all its    preferred embodiment;-   compound (II-I) is selected from the group consisting of compound of    formula (II-1), 1,1′-carbonyldiimidazole,    4-nitrophenylchloroformate, phosgene, diphosgene, triphosgene and    mixtures thereof;

-   step (MIIb) comprises a reaction (MIIb), wherein the reaction    product from the reaction (MIIa) is reacted with a compound of    formula (DRRes-T2-H);-   step (MIIc) comprises a reaction (MIIc), wherein the reaction    product from the reaction (MIIa) is reacted with a compound of    formula (CG3M-II) to provide a compound of formula (IIc);

-   n2 in formula (IIc) is as defined in claim 2;-   step (MIId) comprises a reaction (MIId), wherein compound of formula    (IIc), prepared in reaction (MIIc), is reacted with the compound    (II-I);-   step (MIIe) comprises a reaction (MIIe), wherein the reaction    product from the reaction (MIId) is reacted with a compound of    formula (DRRes-T2-H);-   step (MII0-I-IVa) comprises a reaction (MII0-I-IVa), wherein a    compound of formula (III) is reacted with a compound (II0-I-IVa) to    provide a compound of formula (III0-I-IVa);

-   n2 is 0 in formula (III0-I-IVa);-   compound (II0-I-IVa) is selected from the group consisting of    p-toluenesulfonyl chloride, p-toluenesulfonic anhydride,    methanesulfonyl chloride, methanesulfonic anhydride,    trifluoromethanesulfonyl chloride and trifluoromethanesulfonic    anhydride and mixtures thereof;-   X2 is selected from the group consisting of —OTs, —OMs and —OTf;-   step (MII0-1-IVb) comprises a reaction (MII0-I-IVb), wherein    compound of formula (III0-I-IVa), prepared in reaction (MII0-I-IVa),    is reacted with compound of formula (DRRes-T2-H);-   compound of formula (III) is prepared-   in a step (MIII-IV) for the case that CG1M is a connecting group of    formula (CG1M-IV); or-   in a step (MIII-III) for the case that CG1M is a connecting group of    formula (CG1M-Ill); or-   in two steps, a step (MIII-IIa) and a step (MIII-IIb), or in one    step, a step (MIII-IIc), for the case that CG1M is a connecting    group of formula (CG1M-II); or-   a step (MIII-I) for the case that CG1M is a connecting group of    formula (CG1M-I);-   step (MIII-IV) comprises a reaction (MIII-IV) of a compound of    formula (IV),

-   n2 in formula (IV) is as defined herein, also with all its preferred    embodiments; with a compound of formula (CG1MR-IV);

-   m30 is as defined herein, also with all its preferred embodiments;-   R20 is a residue of formula (R20-1);

-   step (MIII-III) comprises a reaction (MIII-III) of a compound of    formula (IV) with a compound of formula (CG1MR-III);

-   with X1 as defined herein, also with all its preferred embodiments;-   step (MIII-IIa) comprises a reaction (MIII-IIa) of the compound of    formula (IV) with a compound of formula (CG1MR-IIa) to provide a    compound of formula (IV-IIa);

-   with m32 as defined herein, also with all its preferred embodiments;-   step (MIII-IIb) comprises a reaction (MIII-IIb) of compound of    formula (IV-IIa) prepared in step (MIII-IIa) with a compound of    formula (HOSu);

-   step (MIII-IIc) comprises a reaction (MIII-IIc) of the compound of    formula (IV) with a compound of formula (CG1MR-IIc);

-   with m32 as defined herein, also with all its preferred embodiments;-   step (MIII-I) comprises a reaction (MIII-I) of a compound of    formula (IV) with a compound of formula (MA);

-   compound of formula (IV) is prepared in a step (MIV);-   step (MIV) comprises a reaction (MIV), reaction (MIV) is a reduction    of a compound of formula (V) with a compound (IV);

-   compound (IV) is selected from the group consisting of NaBH₄, BH₃,    DIBAL-H, sodium bis(2-methoxyethoxy)aluminium hydride and mixtures    thereof;-   SG, n1, AA, n4, n3, (3), R1 and R2 have the same definition as    above, also with all their preferred embodiments;-   compound of formula (V) is prepared in a step (MVb);-   step (MVb) comprises a reaction (MVb), wherein R30 is cleaved off    from compound of formula (Va) with HCl;

-   R30 is connected to SG via the bond denoted with (**) in the    formulae of SG and is Boc;-   step (MII0-IV) comprises a reaction (MII0-IV) of a compound of    formula (III0) with a compound of formula (CG1MR-IV);

-   with-   SG, CG2, n1, AA, n4, n3, (3), R1, R2, T2 and DRRes as defined    herein, also with all their preferred embodiments, and n2 is 0 in    formula (III0);-   step (MII0-III) comprises a reaction (MII0-III) of a compound of    formula (III0) with a compound of formula (CG1MR-III);-   step (MII0-IIa) comprises a reaction (MII0-IIa) of the compound of    formula (III0) with a compound of formula (CG1MR-IIa) to provide a    compound of formula (II10-IIa);

-   with-   SG, CG2, n1, AA, n4, n3, (3), R1, R2, T2 and DRRes and m32 as    defined herein, also with all their preferred embodiments, and n2 is    0 in formula (III0-IIa);-   step (MII0-11b) comprises a reaction (MII0-IIb) of compound of    formula (III0-IIa) prepared in step (MII0-IIa) with a compound of    formula (HOSu);-   step (MII0-11c) comprises a reaction (MII0-IIc) of the compound of    formula (III0) with a compound of formula (CG1MR-IIc);-   step (MII0-1) comprises a reaction (MII0-1) of a compound of formula    (III0) with a compound of formula (MA);-   compound of formula (III0) is prepared in a step (MIII0),-   step (MIII0) comprises a reaction (MIII0), wherein R30 is cleaved    off from compound of formula (IV0) with HCl;

-   n2 is 0 in formula (IV0);-   compound of formula (IV0) is prepared in a step (MIV0a) and a step    (MIV0b),-   step (MIV0a) comprises a reaction (MIV0a), wherein a compound of    formula (V0) is reacted with a compound (RIV0a) to provide a    compound of formula (IV0a);

-   with-   R30, SG, CG2, n1, AA, n4, n3, (3), R1 and R2 as defined herein, also    with all their preferred embodiments;-   compound (RIV0a) is selected from the group consisting of    p-toluenesulfonyl chloride, p-toluenesulfonic anhydride,    methanesulfonyl chloride, methanesulfonic anhydride,    trifluoromethanesulfonyl chloride and trifluoromethanesulfonic    anhydride, SOCl₂, (COCl)₂, POCl₃, PCl₃, PCl₅, POBr₃, PBr₃, PBr₅,    N-bromosuccinimide, N-iodosuccinimide, HCl, HBr, HI and mixtures    thereof;-   X3 is selected from the group consisting of —OTs, —OMs, —OTf, —C,    —Br and —I;-   step (MIV0b) comprises a reaction (MIV0b), wherein compound of    formula (IV0a), prepared in reaction (MIV0), is reacted with    compound of formula (DRRes-T2-H);-   compound of formula (V0) is prepared in a step (MV0),-   step (MV0) comprises a reaction (MV0), reaction (MV0) is a reduction    of a compound of formula (Va) with a compound (IV);-   CG1M and X1 are as defined herein, also with all its preferred    embodiments;-   SG, n1, n4, n3, AA^(n4), (3), R1, R2, T4, CG3, n2, T2, DRRes,    compound of formula (DRRes-T2-H), m30, m32, R5 and R6 are as defined    herein, also with all its preferred embodiments;-   CG2 is as defined herein, also with all its preferred embodiments.

The compounds (II-I) and of formula (CG3M-II) are known compounds andcan be prepared according to known methods, often they are evencommercially available.

Reaction (MIIa) and reaction (MIId) are similar reactions and can bedone under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Preferably, compound (II-I) is compound of formula (II-1).

Reaction (MIIa) and reaction (MIId) are usually done in a solvent(MIIa).

Preferably, solvent (MIIa) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF and mixtures thereof.

Preferably, the amount of solvent (MIIa) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (III).

Preferably, in the reaction (MIIa) and in the reaction (MIId), from 0.5to 20 mol equivalents, more preferably from 1 to 10 mol equivalents,even more preferably from 1 to 2 mol equivalents, of compound (II-I) areused, the mol equivalents being based the mol of compound of formula(III).

Reaction (MIIa) and reaction (MIId) can be done in the presence of abase (MIIa). Preferably, the base (MIIa) is selected from the groupconsisting of K₂CO₃, Na₂CO₃, diisopropylethylamine, triethylamine,pyridine, 4-dimethylaminopyridine and mixtures thereof.

Preferably, in the reaction (MIIa) and in the reaction (MIId), from 0.5to 50 mol equivalents, more preferably from 1 to 20 mol equivalents,even more preferably from 2 to 10 mol equivalents, of base (MIIa) areused, the mol equivalents being based the mol of compound of formula(III).

Preferably, the reaction (MIIa) and the reaction (MIId) are done underinert atmosphere.

After the reaction (MIIa) and the reaction (MIId), the reaction productof reaction (MIIa) and of reaction (MIId) can be isolated by standardmethods such as washing, extraction, filtration, concentration anddrying. Any of the compounds can be purified before or after isolation,preferably by chromatography or crystallization from an appropriatesolvent.

The crude reaction mixture from reaction (MIIa) and from reaction (MIId)can also be directly used in reaction (MIIb) or reaction (MIIe).

More preferably, reaction (MIIa) and reaction (MIIb) as well as reaction(MIId) and reaction (MIIe) are done consecutively in the same solventand in one pot.

Reaction (MIIb) and reaction (MIIe) are similar reactions and can bedone under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Reaction (MIIb) and reaction (MIIe) are usually done in a solvent(MIIb).

Preferably, solvent (MIIb) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIIb) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of the reaction product of reaction (MIIa) or of reactionproduct of reaction (MIId) respectively.

Preferably, in reaction (MIIb) and in reaction (MIIe), from 0.2 to 10mol equivalents, more preferably from 0.5 to 5 mol equivalents, evenmore preferably from 0.8 to 2 mol equivalents, of compound of formula(DRRes-T2-H) are used, the mol equivalents being based the mol of thereaction product of reaction (MIIa) or of the reaction product ofreaction (MIId) respectively.

Preferably, reaction (MIIb) and reaction (MIIe) are done under inertatmosphere.

Reaction (MII0-I-IVb) and reaction (MIV0b) are similar reactions and canbe done under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Reaction (MII0-I-IVb) and reaction (MIV0b) are usually done in a solvent(MIV0b).

Preferably, solvent (MIV0b) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF and mixtures thereof.

Preferably, the amount of solvent (MIV0b) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (III0-I-IVb) or of formula (IV0a)respectively.

Preferably, in reaction (MII0-I-IVb) and in reaction (MIV0b), from 0.2to 10 mol equivalents, more preferably from 0.5 to 5 mol equivalents,even more preferably from 0.8 to 2 mol equivalents, of compound offormula (DRRes-T2-H) are used, the mol equivalents being based the molof compound of formula (III0-I-IVb) or of formula (IV0a) respectively.

Preferably, reaction (MII0-I-IVb) and reaction (MIV0b) are done underinert atmosphere.

Reaction (MIIc) is usually done in a solvent (MIIc).

Preferably, solvent (MIIc) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIIc) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of reaction product of reaction (MIIa).

Preferably, in the reaction (MIIc) from 0.2 to 20 mol equivalents, morepreferably from 0.5 to 10 mol equivalents, even more preferably from 0.8to 5 mol equivalents, of compound of formula (CG3M-II) are used, the molequivalents being based the mol of the reaction product of reaction(MIIa).

Preferably, the reaction (MIIc) is done under inert atmosphere.

After the reaction (MIIc), the reaction product of the reaction (MIIc)can be isolated by standard methods such as washing, extraction,filtration, concentration and drying. Any of the compounds can bepurified before or after isolation, preferably by chromatography orcrystallization from an appropriate solvent.

After reaction (MIIb), reaction (MIIe), reaction (MII0-I-IVb) andreaction (MIV0b), compound of formula (II) or compound of formula (IV0)respectively can be isolated by standard methods such as washing,extraction, filtration, concentration and drying. Any of the compoundscan be purified before or after isolation, preferably by chromatographyor crystallization from an appropriate solvent.

Preferably, CG1M is a connecting group of formula (CG1M-IV);

more preferably, CG1M is a connecting group of formula (CG1M-IV) and m30is 2.

Compound of formula (V) and compound of formula (III0) can be used inunprotonated form or in protonated form as a salt in reaction (MIV),reaction (MII0-I), reaction (MII0-IIa), reaction (MII0-III) and reaction(MII0-IV)

Reaction (MIII-IV) and reaction (MII0-IV) are similar reactions and canbe done under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Reaction (MIII-IV) and reaction (MII0-IV) are usually done in a solvent(MIII-IV).

Preferably, solvent (MIII-IV) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIII-IV) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (IV).

Preferably, in the reaction (MIII-IV) and in the reaction (MII0-IV),from 1 to 20 mol equivalents, more preferably from 1 to 10 molequivalents, even more preferably from 1 to 5 mol equivalents, ofcompound (CG1MR-IV) are used, the mol equivalents being based the mol ofcompound of formula (IV) or of formula (III0) respectively.

Preferably, reaction (MIII-IV) and reaction (MII0-IV) are done underinert atmosphere.

Reaction (MIII-IV) and reaction (MII0-IV) is usually done in thepresence of a base (MIII-IV).

Preferably, the base (MIII-IV) is selected from the group consisting ofK₂CO₃, Na₂CO₃, diisopropylethylamine, triethylamine, pyridine,4-dimethylaminopyridine and mixtures thereof.

Preferably, in the reaction (MIII-IV) and in reaction (MII0-IV), from0.5 to 50 mol equivalents, more preferably from 1 to 20 mol equivalents,even more preferably from 2 to 10 mol equivalents, of base (MIII-IV) areused, the mol equivalents being based the mol of compound of formula(IV) or of formula (III0) respectively.

After reaction (MIII-IV) and reaction (MII0-IV), the compound of formula(III) or of formula (II) respectively can be isolated by standardmethods such as washing, extraction, filtration, concentration anddrying. The compound of formula (III) or of formula (II) respectivelycan be purified before or after isolation, preferably by chromatographyor crystallization from an appropriate solvent.

Reaction (MIII-III) and reaction (MII0-III) are similar reactions andcan be done under similar reaction parameters ranges as defined herein,with the individual reaction parameters for each of said two reactionsbeing selected independently from each other.

Reaction (MIII-III) and reaction (MII0-Ill) are usually done in asolvent (MIII-III).

Preferably, solvent (MIII-III) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIII-III) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (IV).

Preferably, in reaction (MIII-III) and in reaction (MII0-III), from 1 to20 mol equivalents, more preferably from 1 to 10 mol equivalents, evenmore preferably from 1 to 5 mol equivalents, of compound (CG1MR-III) areused, the mol equivalents being based the mol of compound of formula(IV) or of formula (III0) respectively.

Preferably, reaction (MIII-III) and reaction (MII0-III) are done underinert atmosphere.

After reaction (MIII-III) and reaction (MII0-III), the compound offormula (III) or of formula (II) respectively can be isolated bystandard methods such as washing, extraction, filtration, concentrationand drying. The compound of formula (III) or of formula (II)respectively can be purified before or after isolation, preferably bychromatography or crystallization from an appropriate solvent.

Reaction (MIII-IIa) and reaction (MII0-IIa) are similar reactions andcan be done under similar reaction parameters ranges as defined herein,with the individual reaction parameters for each of said two reactionsbeing selected independently from each other.

Reaction (MIII-IIa) and reaction (MII0-IIa) are usually done in asolvent (MIII-IIa).

Preferably, solvent (MIII-IIa) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIII-IIa) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (IV).

Preferably, in reaction (MIII-IIa) and in reaction (MII0-IIa), from 1 to20 mol equivalents, more preferably from 1 to 10 mol equivalents, evenmore preferably from 1 to 5 mol equivalents, of compound (CG1MR-IIa) areused, the mol equivalents being based the mol of compound of formula(IV) or of formula (III0).

Preferably, reaction (MIII-IIa) and reaction (MII0-IIa) done under inertatmosphere.

After reaction (MIII-IIa) and reaction (MII0-IIa), the compound offormula (IV-IIa) or of formula (III0-IIa) respectively can be isolatedby standard methods such as washing, extraction, filtration,concentration and drying. The compound of formula (IV-IIa) or of formula(III0-IIa) respectively can be purified before or after isolation,preferably by chromatography or crystallization from an appropriatesolvent.

Reaction (MIII-IIb) and reaction (MII0-IIb) are similar reactions andcan be done under similar reaction parameters ranges as defined herein,with the individual reaction parameters for each of said two reactionsbeing selected independently from each other.

Preferably, reaction (MIII-IIb) and reaction (MII0-IIb) are done in thepresence of a compound (COUPADD).

Compound (COUPADD) is a coupling additive conventionally used in peptidechemistry for the coupling reaction of amino acid to peptides by amidebond formation. Preferably, compound (COUPADD) is selected from thegroup consisting of DCC, EDC and mixtures thereof.

Preferably, in reaction (MIII-IIb) and in reaction (MII0-IIb), from 1 to20 mol equivalents, more preferably from 1 to 10 mol equivalents, evenmore preferably from 1 to 5 mol equivalents, of compound (COUPADD) areused, the mol equivalents being based the mol of compound of formula(IV-IIa) or of formula (II0-IIa) respectively.

Reaction (MIII-IIb) and reaction (MII0-IIb) are usually done in asolvent (MIII-IIb).

Preferably, solvent (MIII-IIb) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, TH F and mixtures thereof.

Preferably, the amount of solvent (MIII-IIb) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (IV-IIa).

Preferably, in reaction (MIII-IIb) and in reaction (MII0-IIb), from 1 to20 mol equivalents, more preferably from 1 to 10 mol equivalents, evenmore preferably from 1 to 5 mol equivalents, of compound of formula(HOSu) are used, the mol equivalents being based the mol of compound offormula (IV-IIa) or of formula (III0-IIa) respectively.

Preferably, reaction (MIII-IIb) and reaction (MII0-IIb) are done underinert atmosphere.

After reaction (MIII-IIb) and reaction (MII0-IIb), the compound offormula (III) or of formula (II) respectively can be isolated bystandard methods such as washing, extraction, filtration, concentrationand drying. The compound of formula (III) or of formula (II)respectively can be purified before or after isolation, preferably bychromatography or crystallization from an appropriate solvent.

Reaction (MIII-IIc) and reaction (MII0-IIc) are similar reactions andcan be done under similar reaction parameters ranges as defined herein,with the individual reaction parameters for each of said two reactionsbeing selected independently from each other.

Reaction (MIII-IIc) and reaction (MII0-IIc) are usually done in asolvent (MIII-IIc).

Preferably, solvent (MIII-IIc) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIII-IIc) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (IV).

Preferably, in reaction (MIII-IIc) and in reaction (MII0-IIc), from 1 to20 mol equivalents, more preferably from 1 to 10 mol equivalents, evenmore preferably from 1 to 5 mol equivalents, of compound (CG1MR-IIa) areused, the mol equivalents being based the mol of compound of formula(IV) or of formula (III0) respectively.

Preferably, reaction (MIII-IIc) and reaction (MII0-IIc) are done underinert atmosphere.

Reaction (MIII-IIc) and reaction (MII0-IIc) are usually done in thepresence of a base (MIII-IIc).

Preferably, the base (MIII-IIc) is selected from the group consisting ofK₂CO₃, Na₂CO₃, diisopropylethylamine, triethylamine, pyridine,4-dimethylaminopyridine and mixtures thereof.

Preferably, in the reaction (MIII-IIc) and reaction (MII0-IIc), from 0.5to 50 mol equivalents, more preferably from 1 to 20 mol equivalents,even more preferably from 2 to 10 mol equivalents, of base (MIII-IIc)are used, the mol equivalents being based the mol of compound of formula(IV) or of formula (III0) respectively.

After reaction (MIII-IIc) and reaction (MII0-IIc), the compound offormula (III) or of formula (II) respectively can be isolated bystandard methods such as washing, extraction, filtration, concentrationand drying. The compound of formula (III) or of formula (II)respectively can be purified before or after isolation, preferably bychromatography or crystallization from an appropriate solvent.

Reaction (MIII-I) and reaction (MII0-I) are similar reactions and can bedone under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Preferably, the reaction time of reaction (MIII-I) and of reaction(MII0-1) is from 1 min to 168 h, more preferably from 2 to 144 h, evenmore preferably from 12 to 120 h.

Reaction (MIII-I) and reaction (MII0-I) are usually done in a solvent(MIII-1). Preferably, solvent (MIII-I) is selected from the groupconsisting of N,N-dimethylformamide, N,N-dimethylacetamide,N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF andmixtures thereof.

Preferably, the amount of solvent (MIII-I) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (IV) or of formula (III0)respectively.

Preferably, in reaction (MIII-I) and in reaction (MII0-1), from 1 to 20mol equivalents, more preferably from 1 to 10 mol equivalents, even morepreferably from 1 to 5 mol equivalents, of compound of formula (MA) areused, the mol equivalents being based the mol of compound of formula(IV) or of formula (II0) respectively.

Preferably, reaction (MIII-I) and reaction (MII0-I) are done under inertatmosphere.

Preferably, reaction (MIII-I) and reaction (MII0-I) are done in thepresence of compound (COUPADD).

Preferably, in reaction (MIII-I) and reaction (MII0-I), from 1 to 20 molequivalents, more preferably from 1 to 10 mol equivalents, even morepreferably from 1 to 5 mol equivalents, of compound (COUPADD) are used,the mol equivalents being based the mol of compound of formula (IV) orof formula (III0) respectively.

Preferably, reaction (MIII-I) and reaction (MII0-I) are done in thepresence of compound of formula (HOSu).

Preferably, reaction (MIII-I) and reaction (MII0-I) are done in thepresence of compound (COUPADD) and compound of formula (HOSu).

Preferably, in reaction (MIII-I) and reaction (MII0-I), from 1 to 20 molequivalents, more preferably from 1 to 10 mol equivalents, even morepreferably from 1 to 5 mol equivalents, of compound of formula (HOSu)are used, the mol equivalents being based the mol of compound of formula(IV) or of formula (III0) respectively.

After reaction (MIII-I) and reaction (MII0-I), the compound of formula(III) or of formula (II) respectively can be isolated by standardmethods such as washing, extraction, filtration, concentration anddrying. The compound of formula (III) or of formula (II) respectivelycan be purified before or after isolation, preferably by chromatographyor crystallization from an appropriate solvent.

Reaction (MIV) and reaction (MV0) are similar reactions and can be doneunder similar reaction parameters ranges as defined herein, with theindividual reaction parameters for each of said two reactions beingselected independently from each other.

Preferably, the reaction time of reaction (MIV) and of reaction (MV0) isfrom 1 min to 168 h, more preferably from 1 to 120 h. even morepreferably from 6 to 48 h.

Reaction (MIV) and reaction (MV0) are usually done in a solvent (MIV).

Preferably, solvent (MIV) is selected from the group consisting ofwater, methanol, ethanol, N,N-dimethylformamide, N,N-dimethylacetamide,N,N-dimethylsulfoxide, acetonitrile, acetone, 1,4-dioxane, THF, water,methanol, ethanol and mixtures thereof.

Preferably, the amount of solvent (MIV) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (V) or of formula (Va)respectively.

Preferably, reaction (MIV) and reaction (MV0) are done under inertatmosphere.

-   Preferably, compound (IV) is selected from the group consisting of    NaBH₄, DIBAL-H and mixtures thereof.-   Compound (IV) used in reaction (MIV) and compound (IV) used in    reaction (MV0) can be independently from each identical or    different.

Preferably, in the reaction (MIV) and in reaction (MV0), from 1 to 50mol equivalents, more preferably from 1 to 20 mol equivalents, even morepreferably from 2 to 10 mol equivalents, of compound (IV) are used, themol equivalents being based the mol of compound of formula (V) or offormula (Va).

Reaction (MIV) and reaction (MV0) can be done in the presence of a salt(MIV), salt (MIV) is selected from the group consisting of LiCl, CaCl₂,AlCl₃, ZnCl₂ and mixtures thereof.

Preferably, if salt (MIV) is used in the reaction (MIV) and in reaction(MV0), from 1 to 20 mol equivalents, more preferably from 1 to 10 molequivalents, even more preferably from 1.5 to 5 mol equivalents, of salt(MIV) are used, the mol equivalents being based the mol of compound offormula (V) or of formula (Va).

After reaction (MIV) and reaction (MV0), the compound of formula (IV) orof formula (V0) respectively can be isolated by standard methods such aswashing, extraction, filtration, concentration and drying. The compoundof formula (IV) or of formula (V0) respectively can be purified beforeor after isolation, preferably by chromatography or crystallization froman appropriate solvent.

Reaction (MII0-I-IVa) and reaction (MIV0a) are similar reactions and canbe done under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Preferably, the reaction time of reaction (MII0-I-IVa) and of reaction(MIV0a) is from 1 min to 168 h, more preferably from 2 to 144 h, evenmore preferably from 12 to 120 h.

Reaction (MII0-I-IVa) and reaction (MIV0a) are usually done in a solvent(MIV0a).

Preferably, solvent (MIV0a) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MIV0a) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (V0).

Preferably, in reaction (MII0-I-IVa) and in reaction (MIV0a), from 1 to20 mol equivalents, more preferably from 1 to 10 mol equivalents, evenmore preferably from 1 to 5 mol equivalents, of compound (II0-1-IVa) orof compound (RIV0a) respectively are used, the mol equivalents beingbased the mol of compound of formula (V0).

Preferably, reaction (MII0-I-IVa) and reaction (MIV0a) are done underinert atmosphere.

Preferably, reaction (MII0-I-IVa) and reaction (MIV0a) are done in thepresence of compound of formula (HOSu).

Preferably, in reaction (MII0-I-IVa) and reaction (MIV0a), from 1 to 20mol equivalents, more preferably from 1 to 10 mol equivalents, even morepreferably from 1 to 5 mol equivalents, of compound of formula (HOSu)are used, the mol equivalents being based the mol of compound(II0-I-IVa) or of compound (RIV0a) respectively.

After reaction (MII0-I-IVa) and reaction (MIV0a), compound of formula(III0-I-Va) or of formula (IV0a) respectively can be isolated bystandard methods such as washing, extraction, filtration, concentrationand drying. Any of the compounds can be purified before or afterisolation, preferably by chromatography or crystallization from anappropriate solvent.

-   Further subject of the invention is a method (MVa) for the    preparation of compound of formula (Va), with the compound of    formula (Va) as defined herein, also with all its preferred    embodiments;-   method (MVa) comprises a step (MVa);-   step (MVa) comprises a reaction (MVa), wherein a compound of    formula (VI) is reacted with a compound of formula (SGM);

-   R31 is connected to SG via the bond denoted with (***) in the    formulae of SG and is —OTs, —OMs, —OTf, —Br, —Cl or —I;-   R30 is as defined herein, also with all its preferred embodiments;-   SG, n4, n3, AA^(n4), (3), R1. R2 are as defined herein, also with    all their preferred embodiments.-   Preferably, method (MII) comprises as a further step the step (MVa),    wherein compound of formula (Va) is prepared.

Preferably, the reaction temperature of reaction (MVa) is from 0 to 150°C., more preferably from 20 to 100° C., even more preferably from 30 to60° C.

Preferably, the reaction time of reaction (MVa) is from 1 min to 168 h,more preferably from 1 to 144 h, even more preferably from 12 to 120 h.

Reaction (MVa) is usually done in a solvent (MVa).

Preferably, solvent (MVa) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF and mixtures thereof.

Preferably, the amount of solvent (MVa) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (VI).

Preferably, in the reaction (MVa), from 1 to 20 mol equivalents, morepreferably from 1 to 10 mol equivalents, even more preferably from 1.5to 5 mol equivalents, of compound (SGM) are used, the mol equivalentsbeing based the mol of compound of formula (VI).

Preferably, the reaction (MVa) is done under inert atmosphere.

Reaction (MVa) are usually done in the presence of a base (MVa).

Preferably, base (MVa) is selected from the group consisting of K₂CO₃,Na₂CO₃, diisopropylethylamine, triethylamine, pyridine,4-dimethylaminopyridine and mixtures thereof.

Preferably, in the reaction (MVa), from 1 to 20 mol equivalents, morepreferably from 1 to 10 mol equivalents, even more preferably from 1.5to 5 mol equivalents, of base (MVa) are used, the mol equivalents beingbased the mol of compound of formula (VI).

After the reaction (MVa), compound of formula (Va) can be isolated bystandard methods such as washing, extraction, filtration, concentrationand drying. Any of the compounds can be purified before or afterisolation, preferably by chromatography or crystallization from anappropriate solvent.

Reaction (MVb) and reaction (MIII0) are similar reactions and can bedone under similar reaction parameters ranges as defined herein, withthe individual reaction parameters for each of said two reactions beingselected independently from each other.

Preferably, in reaction (MVb) and in reaction (MIII0), from 1 to 500 molequivalents, more preferably from 5 to 100 mol equivalents, even morepreferably from 10 to 50 mol equivalents, of HCl are used, the molequivalents being based the mol of compound of formula (Va) or offormula (IV0) respectively.

Preferably, the reaction time of reaction (MVb) and of reaction (MIII0)is from 1 min to 168 h, more preferably from 1 to 48 h, even morepreferably from 2 to 24 h.

Reaction (MVb) and reaction (MIII0) is usually done in a solvent (MVb).

Preferably, solvent (MVb) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MVb) is from 1 to 500 fold, morepreferably from 5 to 50 fold, even more preferably from 10 to 30 fold,of the weight of compound of formula (Va) or of formula (IV0).

Preferably, the reaction (MVb) and reaction (MIII0) is done under inertatmosphere.

After reaction (MVb) and reaction (MIII0), compound of formula (V) or offormula (III0) respectively can be isolated by standard methods such aswashing, extraction, filtration, concentration and drying. They can beisolated in protonated form of their salts or in unprotonated form.

Any of the compounds can be purified before or after isolation,preferably by chromatography or crystallization from an appropriatesolvent.

-   Further subject of the invention is a method (MVI) for the    preparation of compound of formula (VI), with the compound of    formula (VI) being as defined herein, also with all its preferred    embodiments;-   method (MVI) comprises a step (MVIa) and optionally a step (MVIb);-   in step (MVIa) the n3 AA^(n4) are consecutively connected to a    compound of formula (VII-1) by peptide coupling reactions and then    to the respective products of the preceding peptide coupling    reactions;

-   step (MVIb) comprises a reaction (MVIb), wherein the N-terminal    amino group of AA denoted with (3) in formula (VI) is reacted with a    compound NTermProt;-   NTermProt is selected from the group consisting of C₁₋₄ alkyl    iodide, C₁₋₄ alkyl bromide, Cl—C(O)-(GRPEG)_(m4)-R3,    R3-C(O)—O—C(O)—R3 and PGNPrec;-   PGNPrec is a reagent which provides for the introduction of PGN on    the N-terminal amino group of AA^(n3) denoted with (3) in formula    (VI);-   GRPEG, m4, R3 and PGN have the same definition as above, also with    all their preferred embodiments;-   n4, n3, AA^(n4) and (3) are as defined herein, also with all their    preferred embodiments.-   Preferably, method (MII) comprises as further steps the step (MVa),    wherein compound of formula (Va) is prepared, and the step (MVIa)    and optionally the step (MVIb), wherein compound of formula (VI) is    prepared.

Compound of formula (VII-1) is a known compound and can be prepared byknown methods.

-   For simplicity sake, AA^(n4) in this text is used both for the    covalently bonded amino acid residue, e.g. in formula (II), and for    the amino acid used in method (MVIa).

In case that AA^(n4) has a side chain with a functional group, thisfunctional group can be protected by a protecting group commonly usedfor protecting functional groups of side chains of amino acids.

Preferably, NTermProt is Ac₂O.

Preferably, PGNPrec is Boc₂O, FmocCl or CbzCl.

Method (MVIa) is done using methodology, parameters and reagentscommonly used in peptide synthesis, and which are known to the skilledperson. Above cited references give the necessary information.

Preferably, the reaction time of reaction (MVIb) is from 1 min to 168 h,more preferably from 1 to 48 h, even more preferably from 2 to 24 h.

Reaction (MVIb) is usually done in a solvent (MVIb).

Preferably, solvent (MVIb) is selected from the group consisting ofN,N-dimethylformamide, N,N-dimethylacetamide, N,N-dimethylsulfoxide,acetonitrile, acetone, 1,4-dioxane, THF, water, methanol, ethanol andmixtures thereof.

Preferably, the amount of solvent (MVIb) is from 1 to 500 fold, morepreferably from 2 to 50 fold, even more preferably from 5 to 20 fold, ofthe weight of compound of formula (VII-1).

More preferably, method (MVIa) and reaction (MVIb) are doneconsecutively in the same solvent.

More preferably, method (MVIa) and reaction (MVIb) are doneconsecutively in the same solvent and in one pot.

Preferably, in the method (MVIa), from 1 to 20 mol equivalents, morepreferably from 1 to 10 mol equivalents, even more preferably from 1 to5 mol equivalents, of NTermProt are used, the mol equivalents beingbased the mol of compound of formula (VII-1).

Preferably, the reaction (MVIb) is done under inert atmosphere.

After the method (MVIa) or reaction (MVIb), the reaction product of themethod (MVIa) or of the reaction (MVIb), each of which is a respectivecompound of formula (VI), can be isolated by standard methods such aswashing, extraction, filtration, concentration and drying. Any of thecompounds can be purified before or after isolation, preferably bychromatography or crystallization from an appropriate solvent.

Compound of formula (SGM) is a known compound and can be preparedaccording to known methods.

Preferably, compound of formula (SGM) is selected from the groupconsisting of compound SGM-II and compound SGM-III.

-   Preferably, compound SGM-II is prepared by reacting a compound of    formula (HSGH-II) first with Boc₂O and then with compound    (SGM-II-R31).-   Compound (SGM-II-R31) is selected from the group consisting of    p-toluenesulfonyl chloride, p-toluenesulfonic anhydride,    methanesulfonyl chloride, methanesulfonic anhydride,    trifluoromethanesulfonyl chloride and trifluoromethanesulfonic    anhydride, SOCl₂, (COCl)₂, POC₃, PCl₃, PCl₅, POBr₃, PBr₃, PBr₅,    N-bromosuccinimide, N-iodosuccinimide, HCl, HBr, HI and mixtures    thereof.

Preferably, compound (SGM-II-R31) is TsCl.H₂N

CH₂

_(m10)

SGPEG

_(m2)OH  (HSGH-II)

-   Preferably, compound SGM-III is prepared by reacting a compound of    formula (HSGH-III) first with Boc₂O and then with a compound of    formula (HSGHReac-1).

-   Compound of formula (HSGH-II) is preferably a compound of formula    (HSGH-II-1) or a compound of formula (HSGH-II-2).

-   Compound SGM-II is preferably a compound of formula (SGM-II-1) or a    compound of formula (SGM-II-2).

-   Compound of formula (HSGH-III) is preferably a compound of formula    (HSGH-III-1) or a compound of formula (HSGH-III-2).

-   Compound SGM-III is preferably a compound of formula (SGM-III-1) or    a compound of formula (SGM-III-2).

Compound of formula (HSGH-II) and compound of formula (HSGH-III) are aknown compound, can be prepared according to known methods, and areoften even commercially available.

-   Compounds of formulae (CG1MR-IV), (CG1MR-III), (CG1MR-IIa), (HOSu)    and compound (COUPADD) are known compounds, can be prepared by known    methods and are often even commercially available.-   Preferably, compound of formula (CG1MR-IV) is prepared by reacting a    compound of formula (CG1MR-IV-OH) with a compound of formula (HOSu);

-   with the compound of formula (HOSu) as defined herein;-   preferably, compound of formula (CG1MR-IV-OH) is prepared by    reacting compound of formula (MA) with compound of formula (AC);

-   with compound of formula (MA) and m30 as defined herein, also with    all their preferred embodiments.

Any of the above defined reactions can be done under similar reactionconditions with the individual reaction parameters for each of thesereactions being selected independently from each other:

-   with respect to pressure: any of the above defined reactions can be    done under vacuum, at atmospheric pressure or even under pressure,    the pressure can for example be up to 10 bar, preferably they are    done under atmospheric pressure;-   with respect to temperature: preferably, the reaction temperature of    any of the above defined reactions is from −20 to 100° C., more    preferably from 0 to 75° C., even more preferably from 10 to 50° C.;-   with respect to reaction time: the reaction time of any of the above    defined reactions is from 1 min to 168 h, more preferably from 0.5    to 24 h, even more preferably from 1 to 12 h;-   if not stated otherwise for any of the above defined reactions.

In particular, compound of formula (I) is selected from the groupconsisting of compound of formula (10), compound of formula (11),compound of formula (12), compound of formula (12-101), compound offormula (13), compound of formula (14), compound of formula (15),compound of formula (15-102) and compound of formula (16);

wherein doxorubicin is the compound of formula (DOXO), which isconnected via the amino group denoted with (d1) in formula (10), (11),(12), (12-101), (13), (14), (15), (15-102) and (16) respectively and informula (DOXO).

In particular, compound of formula (II) is selected from the groupconsisting of compound of formula (20), compound of formula (21),compound of formula (22), compound of formula (23), compound of formula(24), compound of formula (25), compound of formula (26), compound offormula (20-CAMPTO), compound of formula (21-CAMPTO), compound offormula (22-CAMPTO), compound of formula (23-CAMPTO) and compound offormula (21-TAXO-t1-1);

-   wherein-   doxorubicin is the compound of formula (DOXO), which is connected    via the amino group denoted with (d1) in formula (20), (21), (22),    (23), (24), (25) and (26) respectively and in formula (DOXO);-   camptothecin is the compound of formula (CAMPTO), which is connected    via the hydroxy group denoted with (c1) in formula (20-CAMPTO),    (21-CAMPTO), (22-CAMPTO) and (23-CAMPTO) respectively and in formula    (CAMPTO);-   taxo-t1-1 is the compound of formula (TAXO), which is connected via    the hydroxy group denoted with (t1) in formula (21-TAXO-t1-1), in    formula (TAXO-t1-1) and in formula (TAXO).

In particular, compound of formula (IIc) is compound of formula (20c).

In particular, compound of formula (III) is selected from the groupconsisting of compound of formula (30), compound of formula (31),compound of formula (32), compound of formula (33), compound of formula(34), compound of formula (35) and compound of formula (36).

In particular, compound of formula (III0) is compound of formula (300);

-   wherein-   camptothecin is the compound of formula (CAMPTO), which is connected    via the hydroxy group denoted with (c1) in formula (300)    respectively and in formula (CAMPTO).

In particular, compound of formula (CAMPTO).

In particular, compound of formula (III0-I-IVa) is compound of formula(320).

In particular, compound of formula (IV) is selected from the groupconsisting of compound of formula (40), compound of formula (41),compound of formula (42), compound of formula (43), compound of formula(44), compound of formula (45) and compound of formula (46).

In particular, compound of formula (IV0) is compound of formula (400);

-   wherein-   camptothecin is the compound of formula (CAMPTO), which is connected    via the hydroxy group denoted with (c1) in formula (400)    respectively and in formula (CAMPTO).

In particular, compound of formula (IV0a) is compound of formula (400a).

In particular, compound of formula (V) is selected from the groupconsisting of compound of formula (50), compound of formula (50-1),compound of formula (51), compound of formula (51-1), compound offormula (52), compound of formula (52-1), compound of formula (53),compound of formula (53-1), compound of formula (54), compound offormula (54-1), compound of formula (54-2), compound of formula (54-3),compound of formula (55), compound of formula (55-1), compound offormula (56) and compound of formula (56-1).

In particular, compound of formula (V0) is compound of formula (500).

In particular, compound of formula (VI) is selected from the groupconsisting of compound of formula (6), compound of formula (6-1),compound of formula (6-2), compound of formula (6-3), compound offormula (6-4), compound of formula (6b), compound of formula (6b-1),compound of formula (6b-2), compound of formula (6b-3), compound offormula (6b-4), compound of formula (6c) and compound of formula (6-5).

Further subject of the invention is a compound selected from the groupconsisting of compound of formula (I), compound of formula (II),compound of formula (IIc), compound of formula (III), compound offormula (IV), compound of formula (IV-IIa), compound of formula (V),compound of formula (Va), compound of formula (VI), compound of formula(III0-IIa), compound of formula (III0), compound of formula (IV0),compound of formula (IV0a), compound of formula (V0) and compound offormula (III0-I-IVa); with these compounds being as defined herein, alsowith all their preferred embodiments.

Further subject of the invention is a compound selected from the groupconsisting of compound of formula (10), compound of formula (11),compound of formula (12), compound of formula (12-101), compound offormula (13), compound of formula (14), compound of formula (15),compound of formula (15-102), compound of formula (16), compound offormula (20c), compound of formula (20), compound of formula (21),compound of formula (22), compound of formula (23), compound of formula(24), compound of formula (25), compound of formula (26), compound offormula (20-CAMPTO), compound of formula (21-CAMPTO), compound offormula (22-CAMPTO), compound of formula (23-CAMPTO), compound offormula (21-TAXO-t1-1), compound of formula (30), compound of formula(31), compound of formula (32), compound of formula (33), compound offormula (34), compound of formula (35), compound of formula (36),compound of formula (300), compound of formula (320), compound offormula (40), compound of formula (41), compound of formula (42),compound of formula (43), compound of formula (44), compound of formula(45), compound of formula (46), compound of formula (400), compound offormula (400a), compound of formula (50), compound of formula (50-1),compound of formula (51), compound of formula (51-1), compound offormula (52), compound of formula (52-1), compound of formula (53),compound of formula (53-1), compound of formula (54), compound offormula (54-1), compound of formula (54-2), compound of formula (54-3),compound of formula (55), compound of formula (55-1), compound offormula (56), compound of formula (56-1), compound of formula (500),compound of formula (6), compound of formula (6-1), compound of formula(6-2), compound of formula (6-3), compound of formula (6-4), compound offormula (6b), compound of formula (6b-1), compound of formula (6b-2),compound of formula (6b-3), compound of formula (6b-4), compound offormula (6c) and compound of formula (6-5); with these compounds beingas defined herein.

-   Further subject of the invention is the use of the compound of    formula (I), the compound of formula (I) being as defined herein,    also in all its preferred embodiments, for the preparation of a    pharmaceutical composition or of a drug.-   Further subject of the invention is a pharmaceutical composition or    a drug, wherein the pharmaceutical composition and the drug comprise    the compound of formula (I), the compound of formula (I) being as    defined herein, also in all its preferred embodiments.-   Further subject of the invention is a compound of formula (I), a    pharmaceutical composition or a drug, wherein the pharmaceutical    composition and the drug comprise the compound of formula (I), the    compound of formula (I) being as defined herein, also in all its    preferred embodiments,    -   for use in treatment of a disease or an illness, preferably of        cancer.

Compound of formula (II) can be readily covalently attached to a ligandLI. It was surprising, that the protein drug conjugates of the instantinvention, which comprise a connecting group CG2, CG2 being derived fromo-hydrox-p-amino benzylic alcohol, and which comprise a linear peptideresidue, in particular the compounds of formula (I), show increasedplasma stability, and release the drug without the drug being chemicallymodified, Furthermore, they show good water solubility and lowaggregation.

Abbreviations

-   DCM dichloromethane-   DIBAL-H diisobutylaluminum hydride-   DIPEA N,N-diisopropylethylamine-   DMA N,N-dimethylacetamide-   DMAP 4-dimethylaminopyridine-   DMF N,N-dimethyl formamide-   EDTA ethylenediaminetetraacetic acid-   EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline-   ESI-MS electrospray ionisation mass spectrometry-   EtOAc ethyl acetate-   HIC hydrophobic interaction chromatography-   MeCN acetonitrile-   NAP-25 column NAP-25 columns of GE Healthcare are disposable columns    prepacked with Sephadex™ G-25 DNA Grade and require only gravity to    run-   NMR nuclear magnetic resonance-   PE petroleum ether-   Rf retention factor in TLC-   RP-HPLC reversed phase HPLC-   RT room temperature-   SAFC Sigma Aldrich Fine Chemicals-   SEC-HPLC size exclusion chromatography HPLC-   TBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium    tetrafluoroborate-   THF tetrahydrofurane-   TFA Trifluoroacetic acid-   TLC thin layer chromatography    Raw Materials-   compound of formula (DOXO) doxorubicin, commercially available as    hydrochloride salt from Beijing Zhongshuo Pharmaceutical Technology    Development Co. Ltd.-   daunorubicin commercially available as hydrochloride salt from    Aldrich-   PBS The PBS used in the experiments had the composition KH2PO4: 144    mg/L, NaCl: 9000 mg/L and Na2HPO4: 795 mg/L

EXAMPLE 1

To a mixture of p-amino salicylic acid (15.0 g) and MeOH (113.0 ml) at0° C., conc. H₂SO₄ (30.0 ml) was added dropwise. The resulting mixturewas heated to reflux and stirred for 2 hours to form a homogeneoussolution. The reaction mixture was then cooled to RT. Water (360 ml) wasadded, followed by solid NaHCO₃ until pH 7. The resulting mixture wasfiltered, the wet cake was washed with water (3 times with 80 mL each)and dried under vacuum at 55° C. to afford 14.7 g of compound of formula(VII-1) as a solid (89% yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 3.90 (1H, s), 4.12 (2H, brs), 6.16to 6.19 (2H, m), 7.62 to 7.65 (1H, m).

ESI-MS: 168.0 (M+H)+

EXAMPLE 2

To a mixture of H-Cit-OH (40.0 g, 1.0 eq.) and Na₂CO₃ (50.0 g, 2.0 eq.)in water (300 ml) and THF (150 ml), a solution of di-tert-butyldicarbonate (60.0 g, 1.2 eq.) in THF (100 ml) was added dropwise within1 hour. The resulting mixture was allowed to stir at RT overnight. Afterthat, the suspension was washed with PE (2 times with 150 ml each), thenthe mixture was concentrated to about 300 ml under vacuum. The mixturewas acidified to pH 2 with 4.0 M aqueous KHSO₄, then extracted withEtOAc (5 times with 150 ml each). The organic phases were combined andwashed with saturated brine (100 ml), dried over anhydrous Na₂SO₄ andfiltered. The filtrate was evaporated to dryness to afford 52.0 g ofBoc-Cit-OH as white solid (83% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 1.38 (9H, s), 1.36 to 1.43 (2H,m), 1.47 to 1.69 (2H, m), 2.93 (2H, q, J=6.3 Hz), 3.82 to 3.87 (1H, m),5.36 (2H, brs), 5.92 (1H, t, J=5.6 Hz), 7.07 (1H, d, J=8.0 Hz), 12.44(1H, brs).

ESI-MS: 275.8 (M+H)+, 550.8 (2M+H)+

EXAMPLE 3

Boc-Cit-OH (53.0 g, 1.0 eq.), prepared according to example 2, and EEDQ(72.0 g, 1.5 eq.) were added to THF (400 ml). To this mixture was thenadded compound of formula (VII-1) (49.0 g, 1.5 eq.), prepared accordingto example 1. The resulting mixture was stirred at RT for 14 hours. Thenthe reaction mixture was diluted with water (500 ml), extracted withEtOAc (3 times with 250 ml each). The organic phases were combined andwashed with 1.0 M aqueous solution of citric acid (2 time with 150 mleach) and saturated brine (150 ml). After that, the organic phase wasdried over anhydrous Na₂SO₄, then concentrated to dryness, the crudeproduct was purified by silica gel chromatography (eluent PE:EtOAc=4:1to 2:1 to 1:1 (v/v) to pure EtOAc) and isolated to afford 62.5 g ofcompound of formula (6-4) as yellow solid (76% yield).

Analysis by silica gel TLC:EtOAc as eluent (Rf=0.3, UV254)

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.40 (9H, s), 1.57 to 1.83 (4H,m), 3.08 to 3.16 (2H, m), 3.90 (3H, s), 4.43 (1H, s), 5.26 (2H, s), 5.85(2H, s), 7.11 (1H, d, J=8.8 Hz), 7.28 (1H, s), 7.68 (1H, d, J=8.8 Hz),9.74 (1H, s), 10.82 (1H, brs).

ESI-MS: 325.2 (M−tBuOCO+2H)+

EXAMPLE 4

Compound of formula (6-4) (62.0 g, 1 eq.), prepared according to example3, was suspended in a solution of 15% (w/w) HCl in 1,4-dioxane (100 ml),the resulting mixture was stirred at RT for 1 hour. The reaction mixturewas then concentrated under vacuum to afford 51.6 g of compound offormula (6-3) as HCl salt, being a white solid (98% yield).

EXAMPLE 5

Compound of formula (6-3) as HCl salt (75.6 g, 1.0 eq.), preparedaccording to example 4, Boc-L-Val (43.0 g, 1.0 eq.) and TBTU (135.5 g,2.0 eq.) were dissolved in DMF (250 ml). Then DIPEA (71.2 g, 2.6 eq.)was added. The resulting solution was stirred at RT for 17 h. Thereaction mixture was then diluted with water (750 ml), extracted withEtOAc (5 times with 200 ml each), the organic phases were combined andwashed with 1.0 M aqueous NaHCO₃ solution (3 times with 300 ml each) andthen with saturated brine (150 ml). The organic phase was collected andconcentrated to dryness, the crude product was then purified by silicagel chromatography (eluent DCM:MeOH=20:1 to 10:1 to 7:1 (v/v) andisolated to afford 62.0 g of compound of formula (6-2) as a solid (57%yield).

Analysis by silica gel TLC:DCM:MeOH=6:1 (v/v) as eluent (Rf=0.4, UV254)

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.84 (6H, dd, J1=17.2 Hz, J2=6.8Hz), 1.39 (9H, s), 1.42 to 1.51 (2H, m), 1.55 to 1.73 (2H, m), 1.93 to1.98 (1H, m), 2.90 to 3.08 (2H, m), 3.82 to 3.84 (11H, m), 3.88 (3H, s),4.39 to 4.45 (1H, m), 5.43 (2H, s), 6.01 (1H, t, J=5.8 Hz), 6.72 (1H, d,J=8.8 Hz), 7.09 (1H, dd, J1=8.8 Hz, J2=2.0 Hz), 7.73 (1H, d, J=8.8 Hz),8.08 (1H, d, J=7.2 Hz), 10.34 (1H, s), 10.62 (1H, brs).

ESI-MS: 524.3 (M+H)+, 424.3 (M-tBuOCO+2H)+

EXAMPLE 6

Compound of formula (6-2) (62.0 g, 1 eq.), prepared according to example5, was suspended in a solution of 15% (w/w) HCl in 1,4-dioxane (200 ml),the resulting mixture was stirred at RT for 1 hour. The reaction mixturewas then concentrated under vacuum to afford 52.4 g of compound offormula (6-1) as HCl salt, being a white solid (97% yield).

EXAMPLE 7

Compound of formula (6-1) as HCl salt (52.4 g, 1.0 eq.), preparedaccording to example 6, acetic anhydride (60.0 g, 5.0 eq.), pyridine(100.0 g, 11.0 eq.) and methanol (150 ml) were mixed and stirred at RTfor 7 days. The suspension was filtered and the resulting wet cake waswashed with MeOH (4 times with 200 ml each), then dried under vacuum toafford 32.9 g of compound of formula (6) as a white solid (62% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=12.4 Hz, J2=6.8Hz), 1.35 to 1.50 (2H, m), 1.56 to 1.74 (2H, m), 1.87 (3H, s), 1.94 to2.00 (1H, m), 2.93 to 3.06 (2H, m), 3.87 (3H, s), 4.20 (1H, t, J=7.6Hz), 4.34 to 4.39 (1H, m), 5.43 (2H, s), 6.00 (1H, t, J=5.2 Hz), 7.11(1H, dd, J1=8.8 Hz, J2=1.6 Hz), 7.41 (1H, d, J=1.6 Hz), 7.73 (1H, d,J=8.8 Hz), 7.89 (1H, d, J=8.4 Hz), 8.21 (1H, d, J=7.2 Hz), 10.24 (1H,s), 10.62 (1H, s).

ESI-MS: 466.3 (M+H)+, 931.3 (2M+H)+

EXAMPLE 8

Compound of formula (MA) (5.5 g, 1.0 eq.) and beta-alanine (5.0 g, 1.0eq.) in DMF (30.0 ml), were stirred under nitrogen atmosphere for 2hours. The mixture was then cooled to 0° C.

Compound of formula (HOSu) (8.0 g, 1.3 eq.) and DCC (24.0 g, 2.0 eq.)were added. Then the reaction mixture was allowed to warm up to RT andstirred at RT overnight. The reaction mixture was then filtered, theresulting wet cake was washed with DMF (40.0 ml), the organic phaseswere combined and then diluted with water (120 ml) and extracted withDCM (3 times with 50 ml each). The organic phases were combined, washedwith water (50 ml), then with 5% (w/w) aqueous NaHCO3 solution (50 ml)and then with saturated brine (50 ml). The organic phase was dried overanhydrous Na₂SO₄, then concentrated until solid started to precipitate.Then PE (20 ml) was added to the mixture and the resulting mixture wasstirred at RT for 10 min. The mixture was then filtered, the wet cakewas washed with PE (20 ml) and then dried under vacuum at 40° C.overnight to afford 4.0 g of compound of formula (CG1MR-IV-1) as a whitesolid (27% yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 2.84 (4H, s), 3.04 (2H, t, J=7.0Hz), 3.95 (2H, t, J=7.0 Hz), 6.75 (2H, s).

ESI-MS: 267.2 (M+H)+, 289.4 (M+Na)+

EXAMPLE 9

To a mixture of compound of formula (HSGH-II-1) (10.0 g, 1.0 eq.), MeOH(50 ml) and Et3N (10.7 g, 1.1 eq.), a solution of Boc₂O in MeOH (22.8 g,1.1 eq. Boc₂O in 50 ml MeOH) was added dropwise. Then the resultingmixture was stirred at RT for 15 hours and then dried under vacuum toafford 20.0 g of compound of formula (Boc-HSGH-II-1) as a slightlyyellow oil (quantitative yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.44 (9H, s), 2.73 (1H, brs), 3.32(2H, q, J=5.2 Hz), 3.54 to 3.58 (4H, m), 3.72 to 3.74 (2H, m), 5.15 (1H,brs).

EXAMPLE 10

To a mixture of compound of formula (Boc-HSGH-II-1) (6.17 g, 1 eq.),prepared according to example 9, pyridine (2.86 g, 1.2 eq) and DCM (30ml) at 0° C., DMAP (0.366 g, 0.1 eq) were added. A mixture of tosylchloride (6.31 g, 1.1 eq) and DCM (45 ml) was added dropwise. Theresulting mixture was stirred at RT for 8 days. Then the resultingmixture was poured onto DCM (100 ml), the resulting mixture was washedwith water (1 time with 100 ml), the organic phase was then dried overanhydrous Na₂SO₄. The resulting solution was further concentrated andpurified by silica gel chromatography (PE:EtOAc=6:1 to PE:EtOAc=1:1(v/v)) to afford 8.1 g of compound of formula (SGM-II-1) as a colorlessoil (75% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=12.4 Hz, J2=6.8Hz), 1.35 to 1.50 (2H, m), 1.56 to 1.74 (2H, m), 1.87 (3H, s), 1.94 to2.00 (1H, m), 2.93 to 3.06 (2H, m), 3.87 (3H, s), 4.20 (1H, t, J=7.6Hz), 4.34 to 4.39 (1H, m), 5.43 (2H, s), 6.00 (1H, t, J=5.2 Hz), 7.11(1H, dd, J1=8.8 Hz, J2=1.6 Hz), 7.41 (1H, d, J=1.6 Hz), 7.73 (1H, d,J=8.8 Hz), 7.89 (1H, d, J=8.4 Hz), 8.21 (1H, d, J=7.2 Hz), 10.24 (1H,s), 10.62 (1H, s).

EXAMPLE 11

A mixture of compound of formula (6) (3.00 g, 1 eq), prepared accordingto example 7, compound of formula (SGM-II-1) (4.65 g, 2 eq), preparedaccording to example 10, K₂CO₃ (1.82 g, 2 eq) and anhydrous DMF (30 ml)was heated to 50° C. and stirred under nitrogen atmosphere for 7 days.Then the reaction mixture was concentrated to dryness under vacuum.

To the residue methanol (30 ml) was added, the resulting mixture wasstirred for 10 min and then filtered. The wet cake was washed withmethanol (3 times with 10 ml each). The organic phase were combined andthen concentrated. The residue was purified by silica gel columnchromatography (DCM:MeOH=7:1 (v/v)) to afford 3.40 g of compound offormula (50-1) as a slightly yellow solid (81% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) δ 0.86 (6H, dd, J1=12.8 Hz, J2=6.8Hz), 1.38 to 1.51 (2H, m), 1.56 to 1.80 (2H, m), 1.89 (3H, s), 1.92 to2.01 (1H, m), 2.92 to 3.05 (2H, m), 3.07 to 3.12 (2H, m), 3.50 (2H, t,J=6.0 Hz), 3.75 to 3.77 (5H, m), 4.08 to 4.10 (2H, m), 4.20 (1H, dd,J1=8.4 Hz, J2=6.8 Hz), 4.34 to 4.40 (1H, m), 5.42 (2H, s), 5.99 (1H, t,J=5.8 Hz), 6.74 (1H, t, J=5.6 Hz), 7.26 (1H, dd, J1=8.4 Hz, J2=1.6 Hz),7.49 (1H, d, J=2.0 Hz), 7.68 (1H, d, J=8.8 Hz), 7.91 (1H, d, J=8.4 Hz),8.19 (1H, d, J=7.6 Hz), 10.20 (1H, s).

ESI-MS: 653.4 (M+H)+, 675.5 (M+Na)+, 553.5 (M-tBuOCO+2H)+

EXAMPLE 12

A mixture of compound of formula (50-1) (500 mg), prepared according toexample 11, and a solution of 10% (w/w) HCl in 1,4-dioxane (10 ml) wasstirred at RT for 15 hours. The solvent was removed under vacuum toafford 431 mg of compound of formula (50) as HCl salt, being a slightlyyellow solid (quantitative yield).

ESI-MS: 553.5 (M+H)+, 1105.2 (2M+H)+

EXAMPLE 13

To a mixture of compound of formula (50) as HCl salt (1.10 g, 1.0 eq),prepared according to example 12, and anhydrous THF (20 ml) at −30° C.was added a solution of DIBAL-H in hexanes (1 M, 11.9 ml, 6.0 eq). Theresulting mixture was then heated to 0° C. and stirred under nitrogenatmosphere for 15 hours. Then methanol (2.0 mL) was added. Thensaturated aqueous potassium sodium tartrate solution (10 ml) was addedto the mixture and the mixture was stirred for 30 min at RT. Theresulting mixture was evaporated to dryness to generate a white residuewhich was washed with methanol (5 times with 10 ml each). The combinedorganic phases were concentrated and purified by silica gel columnchromatography (DCM:MeOH:Et3N=65:33:2 (v/v)) to afford 0.65 g ofcompound of formula (40) as a white solid (72% yield).

¹H NMR (400 MHz, DMSO-d₆, 60° C.) delta 0.86 (6H, t, J=6.6 Hz), 1.37 to1.53 (2H, m), 1.63 to 1.79 (2H, m), 1.92 (3H, s), 1.99 to 2.07 (1-1, m),2.70 (2H, brs), 2.99 to 3.02 (2H, m), 3.47 (2H, t, J=5.6 Hz), 4.07 (2H,t, J=5.8 Hz), 4.18 (1H, t, J=7.6 Hz), 4.34 to 4.41 (1H, m), 5.25 (2H,s), 5.90 (1H, brs), 7.16 (1H, d, J=8.0 Hz), 7.26 (2H, d, J=8.0 Hz), 7.32(1H, s), 7.76 (1H, d, J=7.6 Hz), 7.91 (1H, d, J=4.4 Hz), 9.69 (1H, s).

ESI-MS: 525.6 (M+H)+

EXAMPLE 14

To a mixture of compound of formula (40) (500.2 mg, 1.0 eq.), preparedaccording to example 13, compound of formula (CG1MR-IV-1) (281.0 mg, 1.1eq), prepared according to example 8, and DMF (9.5 ml) at RT, DIPEA(140.3 mg, 1.1 eq) was added. The resulting mixture was stirred at RTfor 17 hours. Then the DMF was removed under vacuum to afford a slightlyyellow residue. The residue was then mixed with acetone (10 ml) andstirred at RT for 18 h. The mixture was filtered, the wet cake waswashed with acetone (2 times with 5 ml each), then dried under vacuum toafford 515.0 mg of compound of formula (30) as a slightly yellow solid(80% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=11.2 Hz, J2=6.8Hz), 1.32 to 1.48 (2H, m), 1.55 to 1.76 (2H, m), 1.89 (3H, s), 1.94 to2.02 (1H, m), 2.33 (2H, t, J=7.2 Hz), 2.93 to 3.04 (2H, m), 3.16 to 3.20(2H, q, J==5.6 Hz), 3.46 (2H, t, J=5.8 Hz), 3.60 (2H, t, J=7.8 Hz), 3.73(2H, t, J=4.6 Hz), 4.04 (2H, t, J=4.6 Hz), 4.18 (1H, dd, J1=8.4 Hz,J2=6.8 Hz), 4.34 to 4.39 (1H, m), 4.44 (2H, s), 4.88 (1H, brs), 5.42(2H, s), 6.00 (1H, t, J=5.4 Hz), 7.00 (2H, s), 7.16 (1H, dd, J1=8.4 Hz,J2=2.0 Hz), 7.26 (1H, d, J=8.4 Hz), 7.32 (1H, d, J=1.6 Hz), 7.92 (1H, d,J=8.4 Hz), 8.05 (1H, t, J=5.6 Hz), 8.12 (1H, d, J=7.6 Hz), 9.88 (1H, s).

EXAMPLE 15

A mixture of compound of formula (30) (400 mg, 1.0 eq), preparedaccording to example 14, DIPEA (231.0 mg, 3.0 eq), 4 angstrom molecularsieves (800 mg) and dry DMF (8.0 ml) was stirred for 5 min. Thencompound of formula (II-1) (361.2 mg, 2 eq) was added. The resultingmixture was stirred for 3 h at RT. Then compound of formula (DOXO) asHCl salt (342.8 mg, 1.0 eq) was added and the mixture was stirred for 4hours. Then MeCN (40.0 ml) was added. A precipitate had formed and wasfiltered and washed with a mixture of MeCN and DMF (5:1 (v/v), 2 timeswith 5 ml each). The filtrates were combined and dried under vacuum at45° C. to get a dark red residue. The residue was washed with MeCN (2times with 10 ml each), then the residue was dissolved in a mixture ofacetone and water (20:1 (v/v)) and purified by preparative silica gelTLC (DCM:MeOH=5:1 (v/v), Rf=0.15). The product was extracted from thesilica-gel by a mixture of acetone and water (20:1 (v/v), 6 times with20 ml each), the combined extraction solutions were dried under vacuumto afford a crude product as a red solid. The crude product was thenmixed with acetonitrile (5.0 ml), the mixture was stirred at RT for 5hours and then filtered. The presscake was mixed with acetonitrile (5.0ml) and the mixture stirred for 3 hours at RT. The mixture was thenfiltered. The presscake was dried under vacuum at RT to afford 48.0 mgof compound of formula (20) as a red solid (7% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.85 (6H, dd, J1=11.2 Hz, J2=6.8Hz), 1.13 (3H, d, J=6.4 Hz), 1.34 to 1.47 (3H, m), 1.57 to 1.72 (2H, m),1.82 to 1.90 (1H, m), 1.90 (3H, s), 1.93 to 1.98 (1H, m), 2.08 to 2.22(2H, m), 2.29 (2H, t, J=7.2 Hz), 2.91 to 3.01 (4H, m), 3.11 to 3.16 (2H,m), 3.41 (2H, t, J=5.6 Hz), 3.45 to 3.46 (1H, m), 3.54 (2H, t, J=7.2Hz), 3.68 to 3.74 (3H, m), 3.98 (3H, s), 4.03 (2H, t, J=4.0 Hz), 4.17(2H, t, J=7.6 Hz), 4.32 to 4.38 (1H, m), 4.58 (2H, d, J=5.2 Hz), 4.69(2H, d, J=5.6 Hz), 4.85 to 4.89 (2H, m), 4.94 (1H, brs), 5.22 (1H, brs),5.40 (2H, s), 5.46 (1H, brs), 5.98 (1H, t, J=4.8 Hz), 6.80 (1H, d, J=8.0Hz), 6.95 (2H, s), 7.12 (1H, d, J=8.4 Hz), 7.19 (1H, d, J=8.4 Hz), 7.36(1H, s), 7.64 (1H, brs), 7.89 to 7.91 (3H, m), 7.99 (1H, t, J=5.2 Hz),8.10 (1H, d, J=7.2 Hz), 9.92 (1H, s), 13.26 (1H, s), 14.02 (1H, s).

ESI-MS: 1245.5 (M+H)+

EXAMPLE 16

To a mixture of compound of formula (HSGH-III-1) (110.16 g, 5 eq) anddioxane (400 ml) at 0° C., a mixture of Boc₂O (22.10 g, 1 eq) in dioxane(200 ml) was added dropwise. The resulting mixture was then heated to RTand then stirred for 20 hours. Then the solvent was removed undervacuum. The resulting residue was added to water (300 ml), the resultingmixture was extracted with DCM (2 times with 300 ml each). The organiclayer was dried over anhydrous Na₂SO₄ and then evaporated to dryness.The residue was purified by silica gel column chromatography(DCM:EtOAc=1:1 (v/v), then with DCM:MeOH:Et3N=89:9:2 (v/v)) to afford17.7 g of compound of formula (Boc-HSGH-III-1) as a slightly yellow oil(55% yield).

1H NMR (400 MHz, CDCl3, 20° C.) δ 1.44 (9H, s), 1.64 (2H, s), 1.72-1.78(4H, m), 2.81 (2H, t, J=6.6 Hz), 3.23 (2H, q, J=6.0 Hz), 3.55-3.62 (8H,m), 3.64-3.66 (41-H, m), 5.14 (1H, brs).

EXAMPLE 17

To a mixture of compound of formula (Boc-HSGH-III-1) (5.00 g, 1.0 eq),prepared according to example 18, K₂CO₃ (4.30 g, 2.0 eq) and DCM (40 ml)at 0° C., a mixture of compound of formula (HSGHReac-1) (2.17 g, 1.2 eq)in DCM (20 ml) was added dropwise within 1 hour. The resulting mixturewas warmed to RT and stirred for 20 hours. Then the solid was filtered,the cake was washed with DCM (2 times with 5 ml each). The filtrateswere combined and evaporated to dryness. The residue was then purifiedby silica gel column chromatography (eluent EtOAc:PE=3:1) to afford 5.6g of compound of formula (SGM-III-1) as a slightly yellow oil (90%yield).

1H NMR (400 MHz, CDCl3, 20° C.) δ 1.44 (9H, s), 1.64 (2H, s), 1.73-1.86(4H, m), 3.23 (2H, q, J=6.0 Hz), 3.44 (2H, q, J=6.0 Hz), 3.54 (2H, t,J=6.0 Hz), 3.58-3.66 (10H, m), 4.04 (2H, s), 4.98 (1H, brs), 7.31 (1H,brs).

EXAMPLE 18

Compound of formula (6) (5.80 g, 1.0 eq), prepared according to example7, K₂CO₃ (5.18 g, 2.0 eq), compound of formula (SGM-III-1) (9.95 g, 2.0eq), prepared according to example 17, and DMF (45 ml) were mixed. Theresulting mixture was then heated to 50° C. and stirred for 7 days undernitrogen atmosphere. The resulting reaction mixture was evaporated todryness under vacuum. Methanol (40 ml) was added to the residue, theresulting mixture was stirred for 10 min and then filtered. The wet cakewas washed with methanol (3 times with 10 ml each). The organicfiltrates were collected and combined and then evaporated. The residuewas purified by silica gel column chromatography (DCM:MeOH=7:1 (v/v)) toafford 7.20 g of compound of formula (51-1) as a slightly yellow solid(70% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=13.6 Hz, J2=6.8Hz), 1.32 to 1.49 (2H, m), 1.37 (9H, s), 1.55 to 1.78 (6H, m), 1.89 (3H,s), 1.94 to 1.99 (1H, m), 2.93 to 3.07 (4H, m), 3.26 (2H, q, J=6.8 Hz),3.37 (2H, t, J=6.4 Hz), 3.43 to 3.52 (10H, m), 3.81 (3H, s), 4.20 (1H,dd, J1=8.4 Hz, J2=6.8 Hz), 4.35 to 4.41 (1H, m), 4.53 (2H, s), 5.44 (2H,s), 6.03 (1H, t, J=5.6 Hz), 6.74 (1H, t, J=5.6 Hz), 7.36 (1H, dd, J1=8.4Hz, J2=2.0 Hz), 7.41 (1H, d, J=2.0 Hz), 7.80 (1H, d, J=8.8 Hz), 7.90(1H, d, J=8.4 Hz), 8.06 (1H, t, J=5.6 Hz), 8.21 (1H, d, J=7.2 Hz), 10.32(1H, s).

ESI-MS: 726.6 (M-tBuOCO+2H)+, 826.3 (M+H)+, 848.5 (M+Na)+

EXAMPLE 19

Compound of formula (51-1) (1.00 g), prepared according to example 18,and a solution of 15% (v/v) HCl in 1,4-dioxane (10 ml) were mixed. Themixture was stirred at RT for 3 hours. The solvent was removed undervacuum to afford 0.99 g of compound of formula (51) as HCl salt, being aslightly yellow solid (quantitative yield).

ESI-MS: 726.6 (M+H)+

EXAMPLE 20

A mixture of compound of formula (51) as HCl salt (6.08 g, 1 eq),prepared according to example 19, water (100 ml), CaCl₂ (1.86 g, 2.0eq), and NaBH₄ (1.27 g, 4.0 eq) was stirred at RT. Further NaBH₄ wasadded portion wise (1.27 g, 4.0 eq. after a total of 15 hours stirring;1.27 g, 4.0 eq. after a total of 20 hours stirring; 1.27 g, 4.0 eq.after a total of 24 hours of stirring). After a total of 36 hours ofstirring, MeOH (30 ml) was added to the mixture. The reaction mixturewas then filtered and the wet cake was washed with MeOH (3 times with 10ml each). The liquid phase was collected, combined and then evaporatedto dryness. The residue was purified by silica gel column chromatography(eluent DCM:MeOH:Et3N=65:33:2 (v/v)) to afford 2.70 g of compound offormula (41) as a slightly yellow solid (48% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=10.8 Hz, J2=6.8Hz), 1.31 to 1.49 (2H, m), 1.55 to 1.82 (6H, m), 1.90 (3H, s), 1.93 to2.02 (1H, m), 2.82 (2H, t, J=7.4 Hz), 2.98 to 3.05 (2H, m), 3.15 to 3.20(2H, q, J=6.8 Hz), 3.34 to 3.51 (2H, m), 4.16 to 4.20 (1H, m), 4.34 to4.41 (1H, m), 4.46 (2H, s), 4.50 (2H, s), 5.47 (2H, s), 6.13 (1H, t,J=5.6 Hz), 7.25 (2H, s), 7.26 (1H, s), 7.95 (1H, d, J=8.8 Hz), 8.10 (1H,t, J=5.6 Hz), 8.14 (1H, d, J=7.6 Hz), 10.01 (1H, s).

ESI-MS: 698.7 (M+H)+

EXAMPLE 21

Compound of formula (41) (2.78 g, 1.0 eq.), prepared according toexample 20, compound of formula (CG1MR-IV-1) (1.18 g, 1.1 eq), preparedaccording to example 8, and DMF (30 ml) were mixed at RT. Then DIPEA(0.58 g, 1.1 eq) was added. The resulting mixture was stirred at RT for16 hours. Then DMF was removed under vacuum to afford a slightly yellowresidue. The residue was then mixed with acetone (30 ml) and the mixturewas stirred at RT for 5 hours. Then the mixture was filtered, the wetcake was washed with acetone (2 times with 15 ml each) and then driedunder vacuum to afford 2.55 g of compound of formula (31) as a slightlyyellow solid (75% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=11.2 Hz, J2=6.8Hz), 1.32 to 1.48 (2H, m), 1.55 to 1.73 (2H, m), 1.89 (3H, s), 1.93 to2.01 (1H, m), 2.31 (2H, t, J=7.2 Hz), 2.90 to 3.11 (4H, m), 3.17 (2H, q,J=6.4 Hz), 3.36 (2H, t, J=6.4 Hz), 3.44 to 3.60 (8H, m), 3.60 (2H, t,J=7.2 Hz), 4.19 (1H, dd, J1=8.4 Hz, J2=6.8 Hz), 4.35 to 4.40 (1H, m),4.46 (2H, s), 4.51 (2H, s), 5.08 (1H, brs), 5.42 (2H, s), 6.00 (1H, t,J=5.6 Hz), 7.00 (2H, s), 7.22 (1H, s), 7.25 (1H, s), 7.89 to 7.91 (1H,m), 8.04 (1H, t, J=5.6 Hz), 8.10 (1H, d, J=7.6 Hz), 9.92 (1H, s).

ESI-MS: 831.6 (M−OH)+, 849.4 (M+H)+

EXAMPLE 22

A mixture of compound of formula (31) (500.4 mg, 1.0 eq), preparedaccording to example 21, DIPEA (305.7 mg, 4.0 eq), 4 angstrom molecularsieves (500.5 mg) and dry DMF (10.0 ml) was stirred for 5 min. Thencompound of formula (II1-1) (271.1 mg, 1.5 eq.) was added. The resultingmixture was stirred for 5 hours at RT. Then compound of formula (DOXO)as HCl salt (342.8 mg, 1.0 eq.) was added and the resulting mixture wasstirred for 3.5 hours. Then MeCN (50.0 ml) was added. A precipitate hadformed and was filtered and washed with a mixture of MeCN with DMF (5:1(v/v), 3 times with 10 ml each). The filtrates were combined and driedunder vacuum at 45° C. to afford a dark red residue. The residue wasdissolved in a mixture of DCM with MeOH (7:1 (v/v)) and purified bypreparative silica gel TLC (DCM:MeOH=7:1 (v/v), Rf=0.15). The productwas extracted from the silica gel by a mixture of acetone with water(20:1 (v/v), 5 times with 50 ml each), the combined extracts were driedunder vacuum to afford the crude product as a red solid. The crudeproduct was then mixed with acetonitrile (30 ml), the mixture wasstirred at RT for 18 hours and was then filtered. The cake was mixedwith acetonitrile (10 ml) and the mixture was stirred for 3 hours at RT.The mixture was then filtered. The cake was dried under vacuum at RT toafford 100.3 mg of compound of formula (21) as a red solid (12% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.85 (6H, dd, J1=11.8 Hz, J2=6.8Hz), 1.13 (3H, d, J=6.4 Hz), 1.34 to 1.68 (9H, m), 1.82 to 1.90 (1H, m),1.90 (3H, s), 1.93 to 2.00 (1H, m), 2.13 to 2.24 (2H, m), 2.30 (2H, t,J=7.2 Hz), 2.91 to 3.04 (6H, m), 3.10 to 3.15 (2H, m), 3.29 (2H, t,J=6.0 Hz), 3.37 to 3.49 (11H, m), 3.59 (2H, t, J=7.2 Hz), 3.68 to 3.76(III, m), 3.99 (3H, s), 4.13 to 4.19 (2H, m), 4.33 to 4.41 (3H, m), 4.58(2H, d, J=5.6 Hz), 4.71 (1H, d, J=5.2 Hz), 4.86 (1H, t, J=5.8 Hz), 4.95(1H, brs), 5.02 (2H, s), 5.23 (11H, brs), 5.41 (2H, s), 5.46 (1H, brs),5.99 (1H, t, J=4.6 Hz), 6.87 (1H, d, J=7.2 Hz), 7.00 (2H, s), 7.21 to7.25 (3H, m), 7.66 (1H, t, J=4.6 Hz), 8.11 (1H, d, J=7.2 Hz), 9.97 (1H,s), 13.28 (1H, s), 14.04 (1H, s).

ESI-MS: 1417.8 (M+H)+

EXAMPLE 23

To a mixture of compound of formula (HSGH-III-2) (84.9 g, 5.0 eq.) andCHCl₃ (500 ml) was added a mixture of Boc₂O (50.0 g, 1.0 eq.) in CHCl₃(200 ml) dropwise within 2 hours at room temperature. The resultingsolution was stirred for 16 hours at RT. The resulting suspension wasfiltered and the wet cake was washed with DCM (50 ml). The filtrate wascombined and evaporated to dryness to afford a colorless oil. The oilwas then dissolved in DCM (200 ml), washed with water (300 ml) and driedover anhydrous Na₂SO₄. The resulting organic phase was then evaporatedto dryness and the product purified by silica gel column chromatography(eluent DCM:MeOH=20:1 to 5:1 (v/v)) to afford 13.0 g of compound offormula (Boc-HSGH-III-2) (33% yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.32 (9H, s), 1.46-1.53 (4H, m),2.64 (2H, t, J=6.4 Hz), 3.06 to 3.09 (2H, m), 5.22 (1H, brs).

EXAMPLE 24

To a mixture of compound of formula (Boc-HSGH-III-2) (11.3 g), preparedaccording to example 23, K₂CO₃ (18.0 g) and DCM (200 ml) at 0° C. wasadded a mixture of compound of formula (HSGHReac-1) with DCM (50 ml)dropwise within 40 min. The resulting mixture was then heated to RT andstirred for 2 hours. Then aqueous citric acid solution (% by weight,based on the weight of the solution, 180 ml) was added. The organicphase was separated and washed with saturated brine (100 ml), dried overanhydrous Na₂SO₄ and then evaporated to dryness. The residue was thenpurified by silica gel column chromatography (eluent PE:EtOAc2:1 to 1:1to 1:2 (v/v)) to afford 13.9 g of compound of formula (SGM-III-2) as awhite solid (89% yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.46 (9H, s), 1.68 to 1.71 (2H,m), 3.20 (2H, q, J=6.2 Hz), 3.39 (2H, q, J=6.4 Hz), 4.07 (2H, s), 4.87(1H, brs), 7.17 (1H, brs).

EXAMPLE 25

A mixture of compound of formula (6) (5.51 g, 1.0 eq.), preparedaccording to example 7, compound of formula (SGM-III-2) (6.02 g, 2.0eq.), prepared according to example 24, K₂CO₃ (3.31 g, 2.0 eq.), andanhydrous DMF (30 ml) was heated to 50° C. and stirred under nitrogenatmosphere for 7 days. Then the reaction mixture was concentrated todryness under vacuum. MeOH (40 ml) was added to the residue, theresulting mixture was stirred for 10 min and then filtered. The wet cakewas washed with methanol (3 times with 10 ml each). The filtrate wascollected, combined and then concentrated. The residue was purified bysilica gel column chromatography (eluent DCM:MeOH=7:1 (v/v)) to afford3.8 g of compound of formula (52-1) as a slightly yellow solid (46%yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.88 (6H, dd, J1=9.6 Hz, J2=6.8Hz), 1.32 to 1.50 (2H, m), 1.52 to 1.82 (4H, m), 1.90 (3H, s), 1.92 to1.97 (1H, m), 2.91 to 3.05 (4H, m), 3.17 to 3.22 (2H, m), 3.81 (3H, s),4.13 (1H, t, J=7.6 Hz), 4.33 to 4.39 (1H, m), 5.42 (2H, s), 5.98 (1H, t,J=5.8 Hz), 6.80 (1H, 1, J=5.4 Hz), 7.45 (1H, dd, J1=8.6 Hz, J2=1.8 Hz),7.50 (1H, d, J=1.6 Hz), 7.80 (1H, d, J=8.6 Hz), 8.02 to 8.05 (2H, m),8.50 (1H, d, J=11.6 Hz), 10.14 (1H, s).

ESI-MS: 580.5 (M-tBuOCO+2H)+, 680.4 (M+H)+, 1359.0 (2M+H)+

EXAMPLE 26

A mixture of compound of formula (52-1) (3.40 g), prepared according toexample 25, and of a solution of 10% (w/w) HCl in 1,4-dioxane (50 ml)was stirred at RT for 15 hours. Then the reaction mixture wasconcentrated under vacuum to afford 3.20 g of compound of formula (52)as HCl salt, being a white solid (quantitative yield). The crude mixturewas used directly in the next step.

EXAMPLE 27

A mixture of compound of formula (52) as HCl salt (3.00 g, 1 eq.),prepared according to example 26, water (60 ml), CaCl₂ (1.09 g, 2.0 eq.)and NaBH₄ (0.75 g, 4.0 eq.) was stirred at RT. Further amount of NaBH₄was added portionwise (0.75 g, 4.0 eq. after a total of 15 hours ofstirring; 0.74 g, 4.0 eq. after a total of 19 hours of stirring; 0.74 g,4.0 eq. after a total of 23 hours of stirring). After a total of 40hours of stirring, MeOH (15 ml) was added to the mixture. The reactionmixture was then filtered and the wet cake was washed with MeOH (3 timeswith 10 ml each). The filtrate was collected and combined and thenevaporated to dryness. The residue was purified by silica gel columnchromatography (eluent DCM:MeOH:Et3N=65:33:2 (v/v)) to afford 1.05 g ofcompound of formula (42) as a slightly yellow solid (39% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=11.0 Hz, J2=6.8Hz), 1.30 to 1.46 (2H, m), 1.48 to 1.73 (41-, m), 1.89 (3H, s), 1.93 to2.01 (1H, m), 2.56 (1H, t, J=6.8 Hz), 2.91 to 3.06 (4H, m), 3.13 to 3.22(2H, m), 4.16 to 4.20 (1H, m), 4.34 to 4.39 (1H, m), 4.46 (2H, s), 4.50(2H, s), 5.45 (2H, s), 6.06 (1H, t, J=5.6 Hz), 7.22 to 7.27 (3H, m),7.94 (1H, d, J=8.4 Hz), 8.16 to 8.19 (2H, m), 9.96 (1H, s).

ESI-MS: 534.2 (M−OH)+, 552.2 (M+H)+

EXAMPLE 28

Compound of formula (42) (940.4 mg, 1.0 eq.), prepared according toexample 27, compound of formula (CG1MR-IV-1) (501.2 mg, 1.1 eq.),prepared according to example 8, and DMF (10 ml) were mixed. Then DIPEA(247.0 mg, 1.1 eq.) was added at RT. The resulting mixture was stirredat RT for 4 h. Then DMF was removed under vacuum to get a slightlyyellow residue. The residue was then mixed with acetone (20 ml) and theresulting mixture was stirred at RT for 2 hours. Then the mixture wasfiltered, the wet cake was washed with acetone (3 times with 5 ml each)and then dried under vacuum to afford 950.0 mg of compound of formula(32) as a slightly yellow solid (79% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J1=11.2 Hz, J2=6.8Hz), 1.34 to 1.46 (2H, m), 1.47 to 1.73 (4H, m), 1.89 (3H, s), 1.94 to2.01 (1H, m), 2.32 (2H, t, J=7.6 Hz), 2.92 to 3.05 (4H, m), 3.10 to 3.15(2H, m), 3.60 (2H, t, J=7.2 Hz), 4.17 to 4.20 (1H, m), 4.34 to 4.42 (1H,m), 4.46 (2H, s), 4.52 (2H, s), 5.08 (1H, brs), 5.42 (2H, s), 5.99 (1H,t, J=5.6 Hz), 7.00 (2H, s), 7.21 (1H, s), 7.25 (2H, s), 7.90 (III, d,J=8.4 Hz), 7.95 (1H, t, J=5.6 Hz), 8.05 (1H, t, J=5.6 Hz), 8.11 (1H, d,J=7.6 Hz), 9.92 (1H, s).

EXAMPLE 29

A mixture of compound of formula (32) (402.5 mg, 1.0 eq.), preparedaccording to example 28, 4 angstrom molecular sieves (800.0 mg),anhydrous DMF (8.0 ml) and compound of formula (II-1) (347.3 mg, 2.0eq.) was stirred for 5 min. Then DIPEA (297.7 mg, 4.0 eq.) was added.The resulting mixture was stirred for 5 h at RT. Then compound offormula (DOXO) as HCl salt (332.2 mg, 1.0 eq.) was added and the mixturewas then stirred for 4 hours. Then MeCN (40.0 ml) was added. Aprecipitate had formed and was filtered and washed with a mixture ofMeCN and DMF (5:1 (v/v), 2 times with 5 ml each). The filtrate wascombined and dried under vacuum at 45° C. to get a dark red residue. Theresidue was dissolved in a mixture of DCM and MeOH (7:1 (v/v)) andpurified by preparative silica gel TLC (DCM:MeOH=7:1 (v/v), Rf=0.15).The product was extracted from the silica gel by a mixture of acetoneand water (20:1 (v/v), 5 times with 50 ml each), the combined extractionsolutions were dried under vacuum to afford the crude product as a redsolid. The crude product was then mixed with acetonitrile (20 ml), themixture was stirred at RT for 2 hours and then filtered. The cake wasdried under vacuum at RT to afford 74.0 mg of compound of formula (22)as a red solid (10% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.84 (6H, dd, J1=10.8 Hz, J2=6.8Hz), 1.13 (3H, d, J=6.4 Hz), 1.30 to 1.49 (5H, m), 1.56 to 1.69 (2H, m),1.81 to 1.88 (4H, m), 1.93 to 1.98 (1H, m), 2.13 to 2.25 (2H, m), 2.28(2H, t, J=7.2 Hz), 2.92 to 3.07 (8H, m), 3.45 (1H, brs), 3.56 (2H, t,J=7.2 Hz), 3.73 (1H, brs), 3.99 (3H, s), 4.15 to 4.19 (2H, m), 4.35 to4.38 (1H, m), 4.41 (2H, s), 4.58 (2H, d, J=5.8 Hz), 4.71 (1H, d, J=5.6Hz), 4.85 (1H, t, J=6.0 Hz), 4.94 (1H, brs), 5.03 (2H, s), 5.22 (1H,brs), 5.42 (2H, s), 5.47 (1H, brs), 6.02 (1H, t, J=4.6 Hz), 6.87 (1H, d,J=8.0 Hz), 6.97 (2H, s), 7.22 to 7.25 (3H, m), 7.65 (1H, t, J=4.8 Hz),7.88 to 7.90 (5H, m), 8.11 (1H, d, J=7.6 Hz), 9.98 (1H, s), 13.27 (1H,s), 14.02 (1H, s).

ESI-MS: 1271.9 (M+H)+, 1294.4 (M+Na)+

EXAMPLE 30

To a mixture of compound of formula (HSGH-II-2) (21.24 g, 1.0 eq) andEt₃N (21.03 g, 1.1 eq.) in MeOH (100 mL) at 0° C., a solution of Boc₂O(43.83 g, 1.1 eq.) in MeOH (50 ml) was added dropwise within 1 hours.The mixture was then warmed to RT and stirred for 24 hours. Theresulting mixture was then evaporated to dryness under vacuum to afford43.05 g of compound of formula (Boc-HSGH-II-2) as a yellow oil(quantitative yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.30 to 1.43 (4H, m), 1.45 (9H,s), 1.48 to 1.61 (4H, m), 1.70 (1H, brs), 3.12 (2H, q, J=6.8 Hz), 3.64(2H, t, J=6.8 Hz), 4.57 (1H, brs).

EXAMPLE 31

To a mixture of compound of formula (Boc-HSGH-II-2) (20.02 g, 1.0 eq.),prepared according example 30, pyridine (8.02 g, 1.1 eq.) anddichloromethane (50 mL) at 0° C., a mixture of TsCl (19.33 g, 1.1 eq.)in dichloromethane (75 mL) dropwise within 2 hours. The resultingmixture was stirred at RT for 7 days. The solution was then evaporatedto dryness. The residue was washed with a mixture solvent (PE:EtOAc=6:1(v/v), 4 times with 100 ml each) and filtered. The filtrate was combinedthen evaporated to dryness. The crude product was further purified bysilica gel column chromatography (PE:EtOAc=6:1 (v/v)) to afford 14.20 ofcompound of formula (SGM-II-2) as a white solid (41% yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.22 to 1.37 (4H, m), 1.39 to 1.47(11H, ms), 1.62 to 1.68 (2H, m), 2.46 (3H, s), 3.07 (2H, t, J=6.6 Hz),4.02 (2H, t, J=6.4 Hz), 4.52 (1H, brs), 7.36 (2H, d, J=8.0 Hz), 7.78 to7.82 (2H, m).

EXAMPLE 32

A mixture of compound formula (6) (9.31 g, 1.0 eq.), prepared accordingto example 7, compound of formula (SGM-II-2), prepared according toexample 31 (14.90 g, 2.0 eq.), K₂CO₃ (5.65 g, 2.0 eq.) and anhydrous DMF(60 mL) was heated to 50° C. and stirred for under N₂ atmosphere 9 days.The reaction mixture was evaporated to dryness. To this residue MeOH (40mL) was added and, the resulting mixture was stirred for 10 min thenfiltered, the cake was further washed with MeOH (3 times with 15 mleach). The filtrate was combined and evaporated to dryness. The residuewas purified by silica gel column chromatography to afford 6.35 g ofcompound of formula (53-1) as a slight yellow solid (48% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J₁=12.0 Hz, J₂=6.8Hz), 1.24 to 1.33 (2H, m), 1.35 to 1.48 (15H, m), 1.56 to 1.76 (4H, m),1.89 (3H, s), 1.92 to 2.01 (1H, m), 2.89 to 3.05 (4H, m), 3.75 (3H, s),3.97 (2H, t, J=6.2 Hz), 4.17 to 4.21 (1H, m), 4.34 to 4.39 (1H, m), 5.41(2H, s), 5.99 (1H, t, J=5.8 Hz), 6.77 (1H, t, J=5.2 Hz), 7.24 (1H, dd,J=8.6 Hz, J₂=1.8 Hz), 7.47 to 7.48 (1H, m), 7.67 (1H, d, J=8.6 Hz), 7.90(1H, d, J=8.6 Hz), 8.18 (1H, d, J=7.4 Hz), 10.17 (1H, s).

ESI-MS: 565.0 (M−^(t)BuOCO+2H)⁺, 664.8 (M+H)⁺, 1329.1 (2M+H)⁺

EXAMPLE 33

Compound of formula (53-1) (8.10 g), prepared according to example 32,and a solution of 10% (w/w) HCl in 1,4-dioxane (50 ml) were mixed. Themixture was stirred at RT for 1.5 hours. The solvent was removed undervacuum to afford 9.00 g of compound of formula (53) as HCl salt, being awhite solid (quantitative yield).

EXAMPLE 34

To a mixture of compound of formula (53) as HCl salt (4.01 g, 1.0 eq.),prepared according to example 33, and anhydrous THF (40 mL) at −30° C.was added a solution of DIBAL-H in hexanes (1 M, 51.1 ml, 8.1 eq). Theresulting mixture was then heated to 0° C. and stirred under nitrogenatmosphere for 2.5 hours then warmed to RT naturally and stirred for 16hours. The mixture as then cooled to 0° C. Methanol (10 mL) was added.Then saturated potassium sodium tartarate (100 ml) aqueous solution wasadded to the mixture and the mixture was stirred for 1 hour at RT. Theresulting mixture was evaporated to dryness to generate a white residuewhich was mixed with methanol (60 ml) and stirred for 1 hour. Theresulting suspension was filtered, the cake was further washed withmethanol (3 times with 30 ml each). The combined filtrate wasconcentrated and purified by silica gel column chromatography(DCM:Methanol:Et₃N=66:32:2 (v/v)) to afford 1.81 g of compound offormula (43) as a white solid (53% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J₁=10.4 Hz, J₂=6.8Hz), 1.31 to 1.16 (8H, m), 1.55 to 1.74 (4H, m), 1.90 (3H, s), 1.94 to2.02 (1H, m), 2.60 (2H, t, J=6.8 Hz), 2.91 to 3.05 (2H, m), 3.92 (2H, t,J−6.4 Hz), 4.16 to 4.20 (1H, m), 4.34 to 4.39 (1H, m), 4.44 (2H, s),5.43 (2H, s), 6.07 (1H, t, J=5.6 Hz), 7.14 to 7.17 (1H, m), 7.25 (1H, d,J=8.0 Hz), 7.32 (1H, d, J=2.0 Hz), 7.94 (1H, d, J=8.4 Hz), 8.13 (1H, d,J=7.6 Hz), 9.90 (1H, s).

ESI-MS: 519.9 (M−OH)⁺, 538.0 (M+H)⁺, 1129.5 (2M+H)⁺

EXAMPLE 35

A mixture of compound of formula (43) (1.13 g, 1.0 eq), preparedaccording to example 34, maleice anhydride (207.3 mg, 1.1 eq), and DMF(10 mL) was stirred at RT for 20 h. To the resulting solution was addedcompound of formula (HOSu) (225.1 mg, 1.0 eq) and EDC (in its monohydrogen chloride form) (745.0 mg, 2.0 eq). The resulting mixture wasfurther stirred at RT for 4 days. The mixture was evaporated to drynessthen mixed with acetone (30 ml). The resulting mixture was stirred at RTfor 20 h then filtered. The cake was further washed with acetone (3times with 15 ml each) and then dried under vacuum to afford 1.10 g ofcompound of formula (33) as a slightly yellow solid (90% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J₁=10.2 Hz, J₂=6.8Hz), 1.26 to 1.55 (8H, m), 1.59 to 1.78 (4H, m), 1.90 (3H, s), 1.93 to2.02 (1H, m), 2.95 to 3.06 (2H, m), 3.41 (2H, t, J=7.0 Hz), 3.89 (2H, t,J=6.2 Hz), 4.16 to 4.20 (1H, m), 4.34 to 4.40 (1H, m), 4.43 (2H, s),5.42 (2H, brs), 6.05 (1H, brs), 7.00 (2H, s), 7.15 to 7.18 (1H, m), 7.24to 7.29 (2H, m), 7.93 (1H, d, J=8.4 Hz), 8.11 (1H, d, J=7.6 Hz), 9.87(1H, s).

ESI-MS: 599.6 (M−OH)⁺, 639.8 (M+Na)⁺

EXAMPLE 36

A mixture of compound of formula (33) (50.2 mg, 1.0 eq), preparedaccording to example 35, 4 angstrom molecular sieves (100.0 mg),anhydrous DMF (1.0 ml) and compound of formula (II-1) (37.0 mg, 1.6 eq)was stirred at RT for 5 min. Then DIPEA (46.5 mg, 4.4 eq) was added. Theresulting mixture was stirred at RT for 3.5 hours. Then compound offormula (DOXO) as HCl salt (37.8 mg, 0.86 eq) was added and the mixturewas then stirred for 4.5 hours. Then MeCN (5.0 ml) was added. Aprecipitate had formed and was filtered and washed with a mixture ofMeCN and DMF (5:1 (v/v), 2 times with 1 ml each). The filtrate wascombined and dried under vacuum at 45° C. to get a hard residue. Theresidue was dissolved in the mixture of DCM and MeOH (10:1, v/v, 3 ml)and purified by preparative silica gel TLC (DCM:MeOH=7:1, (v/v),Rf=0.35). The product was extracted from the silica gel by a mixture ofacetone and water (20:1, v/v, 6 times with 3 ml each). The combinedextraction solutions were dried under vacuum to afford the crude productas a red solid. The cured product was then mixed with acetonitrile (5ml), the mixture was stirred at RT for 2 hours and then filtered. Thecake was washed with acetonitrile (2 times with 1 ml each) then driedunder vacuum at 25° C. to afford 9.0 mg of compound of formula (23) as ared solid (9% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.84 (6H, dd, J₁=10.2 Hz, J₂=6.8Hz), 1.13 (3H, d, J=6.4 Hz), 1.19 to 1.23 (2H, m), 1.35 to 1.50 (7H, m),1.56 to 1.68 (4H, m), 1.81 to 1.86 (1H, m), 1.88 (3H, s), 1.91 to 2.01(1H, m), 2.11 to 2.23 (2H, m), 2.91 to 3.01 (4H, m), 3.32 (2H, t, J=6.8Hz), 3.45 to 3.47 (1-H, m), 3.69 to 3.74 (1H, m), 3.98 (3H, s), 4.15 to4.19 (2H, m), 4.32 to 4.37 (1H, m), 4.58 (2H, d, J=5.8 Hz), 4.66 (2H, d,J=6.8 Hz), 4.82 to 4.93 (4H, m), 5.23 (1H, brs), 5.39 (2H, s), 5.46 (1H,brs), 5.96 (1H, t, J=5.6 Hz), 6.74 (1H, d, J=8.0 Hz), 6.93 (2H, s), 7.10to 7.18 (2H, m), 7.31 (III, brs), 7.62 to 7.65 (11-1, m), 7.89 to 7.90(3H, m), 8.09 (1H, d, J=7.4 Hz), 9.88 (1H, s), 13.26 (1H, s), 14.02 (1H,s).

ESI-MS: 1185.8 (M+H)⁺, 1208.4 (M+Na)⁺

EXAMPLE 37

A mixture of Z-L-Val (1.47 g, 0.95 eq), anhydrous DMF (15 ml), TBTU(2.97 g, 1.5 eq) and DIPEA (2.00 g, 2.5 eq) was stirred at RT for 15min. To the resulting mixture was added compound of formula (6-3) as HClsalt (2.23 g, 1.0 eq), prepared according to example 4. The mixture wasfurther stirred at RT for 14 hours then evaporated to dryness at 45° C.Water (150 ml) was then added, the resulting mixture was extracted withEtOAc (3 times with 200 ml each). The combined organic phase was washedwith 250 ml saturated brine then dried over anhydrous Na₂SO₄. Theresulting solution was concentrated then purified by silica gel columnchromatography) (PE:EA=1:2 to DCM:MeOH=10:1 (v/v)) to afford 2.92 g ofcompound of formula (6-5) as a white solid (85%) yield.

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.87 (6H, dd, J₁=17.6 Hz, J₂=6.8Hz), 1.36 to 1.50 (2H, m), 1.58 to 1.74 (2H, m), 1.94 to 2.02 (1H, m),2.91 to 3.08 (2H, m), 3.87 (3H, s), 3.90 to 3.96 (1H, m), 4.37 to 4.43(1H, m), 5.04 (2H, s), 5.43 (2H, s), 5.99 (1H, t, J=5.6 Hz), 7.10 (1H,dd, J₁=8.8 Hz, J₂=2.0 Hz), 7.30 to 7.37 (6H, m), 7.41 (1H, d, J=2.0 Hz),7.74 (1H, d, J=8.8 Hz), 8.19 (1H, d, J=7.2 Hz), 10.32 (1H, s), 10.62(1H, s).

ESI-MS: 557.6 (M+H)⁺, 579.9 (M+Na)⁺, 1114.8 (2M+H)⁺, 1136.5 (2M+Na)

EXAMPLE 38

A mixture of compound of formula (6-5) (20.00 g, 1.0 eq), preparedaccording to example 37, K₂CO₃ (9.91 g, 2.0 eq) and anhydrous DMF (80ml). The resulting mixture was heated to 50° C. and stirred for 0.5hour. Compound of formula (SGM-II-1) (25.79 g, 2.0 eq), preparedaccording to example 10, was added to the reaction mixture in 4 portionswithin 2 hours. The reaction mixture was stirred at 50° C. for 3 daysthen evaporated to dryness at 45° C. The residue was washed with MeOH (4times with 20 ml each). The filtrate was combined then concentrated intodryness. The residue was further purified by silica gel columnchromatography (DCM:MeOH=20:1, v/v) to afford 22.17 g of compound offormula (54-1) as a white solid (83% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.87 (61, dd, J=17.6 Hz, J₂=6.8Hz), 1.36 to 1.49 (11H, m), 1.59 to 1.73 (2H, m), 1.96 to 2.03 (1H, m),2.92 to 3.14 (41-, m), 3.50 (2, t, J=6.0 Hz), 3.74 to 3.76 (5H, m), 3.93to 3.97 (1H, m), 4.08 (2H, t, J=4.6 Hz), 4.39 to 4.44 (1H, m), 5.05 (2H,s), 5.42 (2H, s), 5.98 (1H, t, J=5.6 Hz), 6.73 (1H, t, J=5.4 Hz), 7.25(1H, dd, J₁=8.6 Hz, J₂=1.56 Hz), 7.30 to 7.36 (6H, m), 7.48 (1H, s),7.69 (1H, d, J=8.6 Hz), 8.15 (1H, d, J=7.2 Hz), 10.28 (11H, s).

ESI-MS: 745.0 (M+H)⁺, 1488.8 (2M+H)+, 645.3 (M−^(t)BuOCO+2H)⁺

EXAMPLE 39

A mixture of compound of formula (54-1) (4.80 g, 1.0 eq), preparedaccording to example 38, methanol (200 ml) and Pd/C (5%, 0.24 g) wasdegassed for three times then stirred under H₂ atmosphere (6 bar) at 30°C. for 18 hours. The resulting mixture was filtered, the filtrate wasconcentrated to dryness under vacuum to afford 3.96 g of compound offormula (54-2) as a white solid (quantitative yield.)

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.81 (3H, d, J=6.8 Hz), 0.90(3H, d, J=6.8 Hz), 1.36 to 1.49 (11H, m), 1.58 to 1.78 (2H, m), 1.91 to1.99 (1H, m), 2.92 to 3.12 (5H, m), 3.50 (2H, t, J=6.0 Hz), 3.75 to 3.77(5H, m), 4.09 (2H, t, J=4.8 Hz), 4.47 (1H, s), 5.45 (2H, s), 6.07 (1H,t, J=5.6 Hz), 6.72 (1H, t, J=5.2 Hz), 7.24 (1H, dd, J₁=8.6 Hz, J₂=1.6Hz), 7.51 (1H, d, J=1.4 Hz), 7.69 (1H, d, J=8.6 Hz), 8.24 (1H, d, J=8.6Hz), 10.42 (1H, s).

ESI-MS: 611.8 (M+H)⁺, 1221.4 (2M+Na)⁺, 512.0 (M−^(t)BuOCO+2H)⁺

EXAMPLE 40

A mixture of N, N-dimethylglycine (0.48 g, 1.0 eq), DMF (15 ml) and TBTU(3.01 g, 2.0 eq) was cooled to 0° C. The resulting mixture was thenstirred for 15 min followed by addition of compound of formula (54-2)(2.86 g, 1.0 eq), prepared according to example 39. The mixture was thenwarmed to RT naturally and further stirred for 18 hours. The resultingmixture was then evaporated to dryness. The resulting residue waspurified by silica gel column chromatography (DCM:MeOH=7:1, (v/v)) toafford 2.78 g of compound of formula (54-3) as a white solid (85%yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.82 (3H, d, J=6.8 Hz), 0.89(3H, d, J=6.8 Hz), 1.36 to 1.49 (11H, m), 1.56 to 1.75 (5H, m), 1.98 to2.06 (1H, m), 2.33 (6H, s), 2.92 to 3.04 (2H, m), 3.06 to 3.11 (4H, m),3.50 (2H, t, J=6.0 Hz), 3.74 to 3.77 (5H, m), 4.09 (2H, t, J=4.6 Hz),4.30 to 4.34 (1H, m), 4.36 to 4.41 (1H, m), 5.43 (2H, s), 6.01 (1H, t,J=5.8 Hz), 6.73 (1H, t, J=5.2 Hz), 7.25 (1H, dd, J₁=8.6 Hz, J₂=1.8 Hz),7.28 to 7.32 (1H, m), 7.35 to 7.39 (1H, m), 7.46 (1H, d, J=1.6 Hz), 7.55(1H, d, J=8.4 Hz), 7.68 (1H, d, J=8.6 Hz), 7.82 (1H, d, J=9.0 Hz), 7.85to 7.87 (1H, m), 8.38 (1H, d, J=7.2 Hz), 10.30 (1H, s).

ESI-MS: 696.2 (M+H)⁺, 1391.1 (2M+H)⁺, 596.3 (M−^(t)BuOCO+2H)⁺

EXAMPLE 41

A mixture of compound of formula (54-3), prepared according to example40, and a solution of 10% (w/w) HCl in 1,4-dioxane (50 ml) was stirredat RT for 18 hours. The solvent was removed under vacuum to afford 5.60g of compound of formula (54) as di-HCl salt, being a white solid(quantitative yield).

ESI-MS: 596.4 (M+H)⁺, 1191.3 (2M+H)⁺

EXAMPLE 42

To a mixture of compound of formula (54) as di-HCl salt (1.00 g, 1.0eq), prepared according to example 41 and anhydrous THF (15 ml) was at0° C. was added a solution of DIBAL-H in hexanes (1 M, 10 ml, 6.7 eq).The resulting mixture was further at 0° C. for 1.5 hours. Then methanol(10 ml) was added dropwise. Then saturated potassium sodium tartarateaqueous solution (30 mL) was added and stirred at RT for 1 hour. Theresulting mixture was evaporated to dryness. The resulting residue waswashed with MeOH (3 times with 10 ml each). The combined filtrate wasconcentrated then purified by silica gel column chromatography(DCM:MeOH:Et₃N=65:33:2 (v/v)) to afford 0.43 g of compound of formula(44) as a white solid (yield 51%).

¹H NMR (400 MHz. DMSO-d₆, 60° C.) delta 0.82 (3H, d, J=6.8 Hz), 0.87(3H, d, J=6.8 Hz), 1.32 to 1.50 (2H, m), 1.55 to 1.74 (2H, m), 1.97 to2.06 (1H, m), 2.23 (6H, s), 2.73 (2H, t, J=5.6 Hz), 2.91 (2H, brs), 2.93to 3.06 (2H, m), 3.50 (2H, t, J=5.6 Hz), 3.75 (2H, t, J=4.6 Hz), 4.05 to4.07 (2H, m), 4.29 to 4.33 (1H, m), 4.35 to 4.40 (1H, m), 4.45 (2H, s),5.41 (2H, s), 6.02 (1H, t, J=5.6 Hz), 7.15 (III, dd, J₁=8.4 Hz, J₂ 1.6Hz), 7.26 (1H, d, J=8.0 Hz), 7.31 (1H, d, J=1.6 Hz), 7.63 (1H, d, J=9.2Hz), 8.31 (1H, d, J=7.4 Hz), 9.97 (1H, s).

ESI-MS: 550.3 ((M−OH⁻)⁺, 568.2 (M+H)⁺, 1134.7 (2M+H)⁺

EXAMPLE 43

To a mixture of compound of formula (44) (1.10 g, 1.0 eq), preparedaccording to example 42, compound of formula (CG1MR-IV-1) (0.57 g, 1.1eq), prepared according to example 8, and DMF (4.0 ml) at RT, DIPEA(0.28 g, 1.1 eq) was added. The resulting mixture was stirred at RT for18 hours. Then the DMF was removed under vacuum to afford a slightlyyellow residue which was then mixed with acetone (20 ml) and stirred atRT for 18 hours. The mixture was filtered, the cake was washed withacetone (2 times with 5 ml each), then dried under vacuum to afford 1.11g of compound of formula (34) (80% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.82 (3H, d, J=6.8 Hz), 0.88(3H, d, J=6.8 Hz), 1.34 to 1.46 (2H, m), 1.55 to 1.73 (2H, m), 1.97 to2.05 (11-1, m), 2.25 (6H, s), 2.34 (2H, t, J=7.0 Hz), 2.95 to 3.06 (4H,m), 3.17 to 3.21 (2H, m), 3.46 (2H, t, J=5.8 Hz), 3.60 (2H, t, J=7.2Hz), 3.73 (2H, t, J=4.4 Hz), 4.03 (2H, t, J=4.4 Hz), 4.29 to 4.33 (1H,m), 4.35 to 4.40 (1H, m), 4.45 (2H, s), 4.86 (1H, brs), 5.41 (2H, s),5.99 (1H, t, J=5.6 Hz), 7.00 (2H, s), 7.14 to 7.16 (1H, m), 7.26 to 7.30(2H, m), 7.67 (1H, d, J=9.2 Hz), 8.03 (1H, t, J=5.2 Hz), 8.30 (1H, d,J=7.4 Hz), 9.95 (1H, s).

ESI-MS: 701.3 (M−OH⁻)⁺, 719.2 (M+H)⁺, 1436.7 (2M+H)⁺

EXAMPLE 44

A mixture of compound of formula (II-1) (169.4 mg, 2.0 eq), anhydrousDMF (4.0 ml) and 4 angstrom molecular sieves (400.0 mg) was stirred atRT for 15 min. To the resulting mixture was added DIPEA (107.9 mg, 3.0eq.) and compound of formula (34), (200.0 mg, 1.0 eq), preparedaccording to example 43. The resulting mixture was further stirred for2.5 hours. Then compound of formula (DOXO) as HCl salt (161.8 mg, 1.0eq) was added and further stirred for 2 hours. Then MeCN (20 ml) wasadded. A precipitate had formed and was filtered and washed with amixture of MeCN and DMF (5:1 (v/v), 3 times with 3 ml each). Thefiltrates were combined and dried under vacuum at 48° C. The resultingresidue was dissolved in a mixture of DCM and MeOH (5:1 (v/v), 5 ml) andpurified by preparative silica gel TLC (DCM:MeOH=4:1 (v/v), Rf=0.5). Theproduct was extracted from the silica-gel by a mixture of acetone andwater (7:1 (v/v), 6 times with 10 ml each). The combined extractionsolutions were dried under vacuum to afford a crude product as a redsolid. The crude product was then mixed with acetonitrile (5 mL),stirred at RT for 2 hours then filtered. The wet cake was dried undervacuum at RT to afford 7.0 mg of compound of formula (24) as a red solid(2% yield.)

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.80 (3H, d, J=6.8 Hz), 0.86(3H, d, J=6.6 Hz), 1.13 (3H, d, J=6.4 Hz), 1.32 to 1.52 (3H, m), 1.57 to1.69 (2H, m), 1.82 to 1.88 (1H, m), 1.95 to 2.04 (1H, m), 2.10 to 2.18(2H, m), 2.24 (6H, s), 2.29 (2H, t, J=7.4 Hz), 2.92 to 3.02 (6H, m),3.12 to 3.16 (2H, m), 3.41 (2H, t, J=5.6 Hz), 3.45 (1H, brs), 3.55 (2H,t, J=7.2 Hz), 3.69 to 3.74 (3H, m), 3.99 (3H, s), 4.03 (2H, brs), 4.13to 4.17 (1H, m), 4.28 to 4.31 (1H, m), 4.33 to 4.38 (1H, m), 4.58 (2H,d, J=5.2 Hz), 4.69 (1H, d, J=5.6 Hz), 4.86 (1H, t, J=6.0 Hz), 4.90 (1H,s), 4.95 (1H, t, J=4.4 Hz), 5.23 (1H, brs), 5.40 (2H, s), 5.47 (1H, s),6.00 (1H, t, J=5.6 Hz), 6.79 (1H, d, J=8.0 Hz), 6.95 (2H, s), 7.12 (1H,d, J=8.2 Hz), 7.19 (1H, d, J=8.2 Hz), 7.34 (1H, s), 7.64 to 7.68 (2H,m), 7.91 (2H, d, J=4.8 Hz), 7.98 (1H, t, J=5.2 Hz), 8.30 (1H, d, J==7.2Hz), 10.02 (1H, s), 13.27 (1H, s), 14.02 (1H, s).

ESI-MS: 1288.3 (M+H)⁻

EXAMPLE 45

A mixture of compound of formula (VII-1) (26.10 g, 1.5 eq), preparedaccording to example 1, THF (150 ml), Boc-L-Lys(Ac)—OH (30.00 g, 1.0 eq)and EEDQ (51.45 g, 2.0 eq) was stirred at RT for 24 hours. The mixturewas then evaporated to dryness. The residue was purified by silica gelcolumn chromatography (PE:EtOAc=2:1 (v/v) to pure EtOAc) to afford 26.00g of compound of formula (6b-4) as a slightly yellow solid (57% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 1.21 to 1.34 (4H, m), 1.38 (9H,s), 1.58 to 1.66 (2H, m), 1.77 (3H, s), 3.00 to 3.04 (2H, m), 3.87 (3H,s), 3.99 to 4.07 (1H, m), 7.08 to 7.11 (2H, m), 7.40 (1H, d, J=2.0 Hz),7.74 (1H, d, J=8.8 Hz), 7.78 to 7.81 (1H, m), 10.20 (1H, s), 10.62 (1H,s).

ESI-MS: 437.9 (M+H)⁺, 460.3 (M+Na)⁺, 875.1 (2M+H)⁺, 896.9 (2M+Na)⁺,338.1 (M−^(t)BuOCO+2H)⁺

EXAMPLE 46

Compound of formula (6b-4) (26.00 g), prepared according to example 45,was suspended in a solution of 10% (w/w) HCl in 1,4-dioxane (150 ml),the resulting mixture was stirred at RT for 3 hours. The reactionmixture was then concentrated under vacuum to afford 22.05 g of compoundof formula (6b-3) as HCl salt, being a yellow solid (quantitativeyield).

ESI-MS: 339.2 (M+H)⁺

EXAMPLE 47

A mixture of compound of formula (6b-3) as HCl salt (25.21 g, 1.0 eq),prepared according to example 46, Boc-L-Val-OH (13.25 g, 1.0 eq), TBTU(23.66 g, 1.1 eq), DIPEA (19.05 g, 2.2 eq) and DMF (100 ml) was stirredat RT for 18 hours. The resulting mixture was evaporated to dryness thenpurified by silica gel chromatography (DCM:MeOH=10:1 to 7:1, (v/v)) toafford 30.80 g of compound of formula (6b-2) as a slightly yellow solid(90% yield).

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.39 to 1.46 (14H, m), 1.49 to1.56 (2H, m), 1.74 to 1.83 (1H, m), 1.95 to 2.02 (4H, m), 3.17 to 3.30(2H, m), 3.92 (3H, s), 4.17 to 4.22 (1H, m), 4.54 to 4.60 (1H, m), 5.53(1H, brs), 6.32 (1H, brs), 7.15 (1H, d, J=8.0 Hz), 7.31 (1H, brs), 7.38(1H, s), 7.72 (1H, dd, J₁=8.8 Hz, J₂=1.0 Hz), 9.38 (1H, brs), 10.80 (1H,s).

ESI-MS: 509.3 (M+H)⁺, 1017.0 (2M+H)⁺, 1038.8 (2M+Na)⁺, 409.5(M−^(t)BuOCO+2H)⁺

EXAMPLE 48

Compound of formula (6b-2) (30.80 g), prepared according to example 47,was suspended in a solution of 10% (w/w) HCl in 1,4-dioxane (150 ml),the resulting mixture was stirred at RT for 23 hours. The reactionmixture was then concentrated under vacuum to afford 25.90 g of compoundof formula (6b-1) as HCl salt, being a white solid (96% yield).

ESI-MS: 409.0 (M+H)⁺

EXAMPLE 49

A mixture of compound of formula (6b-1) as HCl salt (15.32 g, 1.0 eq),prepared according to example 48, methanol (100 ml), pyridine (9.0 ml,3.2 eq) and acetic anhydride (14.0 ml, 4.3 eq) was stirred at RT for 60hours. The resulting mixture was evaporated to dryness under vacuum. Theresidue was purified by silica gel column chromatography (DCM:MeOH=15:1to 7:1, (v/v)) to afford 12.51 g of compound of formula (6b) as aslightly yellow solid (80% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 1.21 (3H, d, J=7.0 Hz), 1.29 to1.38 (4H, m), 1.61 to 1.74 (2H, m), 1.78 (3H, s), 1.86 (3H, s), 2.99 to3.04 (2H, m), 3.87 (3H, s), 4.27 to 4.34 (2H, m), 7.14 (1H, d, J=8.8Hz), 7.42 (1H, brs), 7.73 (1H, d, J=8.6 Hz), 7.81 (1H, t, J=5.2 Hz),8.09 (1H, d, J=7.0 Hz), 10.18 (1H, s), 10.62 (1H, s).

ESI-MS: 451.3 (M+H)⁺, 473.6 (M+Na)⁺, 900.9 (2M+H)⁺, 923.1 (2M+Na)⁺

EXAMPLE 50

A mixture of compound of formula (6b) (6.73 g, 1.0 eq), preparedaccording to example 49, compound of formula (SGM-II-1) (11.50 g, 2.1eq), prepared according to example 10, K₂CO₃ (4.40 g, 2.1 eq.) andanhydrous DMF (60 ml) was heated to 0° C. and stirred under nitrogenatmosphere for 8 days. The resulting mixture was evaporated to dryness,the residue was washed with MeOH (3 times with 15 ml each). Thefiltrates were combined and evaporated to dryness. The residue waspurified by silica gel column chromatography (DCM:MeOH=15:1 to 7:1,(v/v)) to afford 7.64 g of compound of formula (55-1) as a white solid(81% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 1.21 (3H, d, J=7.2 Hz), 1.25 to1.40 (13H, m), 1.59 to 1.74 (2H, m), 1.78 (3H, s), 1.87 (3H, s), 2.99 to3.04 (2H, m), 3.08 to 3.12 (2H, m), 3.50 (2H, t, J=6.0 Hz), 3.75 to 3.77(5H, m), 4.09 (2H, t, J=4.6 Hz), 4.24 to 4.34 (2H, m), 6.74 (1H, t,J=5.4 Hz), 7.28 (1H, dd, J₁=8.6 Hz, J₂=1.6 Hz), 7.51 (1H, d, J=1.6 Hz),7.69 (1H, d, J=8.6 Hz), 7.81 (1H, t, J=5.2 Hz), 8.08 to 8.13 (2H, m),10.14 (1H, s).

ESI-MS: 638.2 (M+H)⁺, 660.4 (M+Na)⁺, 1275.0 (2M+H)⁺, 538.4(M−^(t)BuOCO+2H)⁺

EXAMPLE 51

Compound of formula (55-1) (7.60 g), prepared according to example 50,was suspended in 10% (w/w) HCl in 1,4-dioxane (50 ml), the resultingmixture was stirred at RT for 5 hours. The reaction mixture was thenconcentrated under vacuum to afford 6.72 g of compound of formula (55)as HCl salt, being a white solid (98% yield).

ESI-MS: 538.5 (M+H)⁺, 1074.8 (2M+H)⁺, 1097.0 (2M+Na)⁺

EXAMPLE 52

To a mixture of compound of formula (55) as HCl salt (6.00 g, 1.0 eq.),prepared according to example 51, and anhydrous THF (50 mL) at 0° C. wasadded a solution of DIBAL-H in hexanes (1M, 63.0 ml, 6.0 eq). Theresulting mixture was then heated to 0° C. and stirred under nitrogenatmosphere for 6 hours. Then methanol (10 mL) was added. After that,saturated potassium sodium tartarate (150 ml) aqueous solution was addedto the mixture and the mixture was stirred at RT for 15 hours. Theresulting mixture was evaporated to dryness to generate a white residuewhich was further washed with methanol (3 times with 50 ml each). Thefiltrates were combined then concentrated, and purified by silica gelcolumn chromatography (DCM:Methanol:Et₃N=75:25:2 (v/v)) to afford 3.27 gof compound of formula (45) as a white solid (61% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 1.22 (3H, d, J=4.4 Hz), 1.27 to1.41 (4H, m), 1.62 to 1.78 (2H, m), 1.79 (3H, s), 1.88 (3H, s), 2.68(2H, t, J=5.6 Hz), 2.99 to 3.03 (2H, m), 3.46 (2H, t, J=5.6 Hz), 3.73(2H, t, J=4.4 Hz), 4.04 (2H, t, J=4.4 Hz), 4.21 to 4.38 (2H, m), 4.45(2H, s), 5.36 (1H, s), 7.22 to 7.28 (2H, m), 7.40 (1H, s), 8.00 (1H, t,J=5.4 Hz), 8.16 (1H, d, J=8.0 Hz), 8.27 (1H, d, J=6.8 Hz), 10.05 (1H,s).

ESI-MS: 510.5 (M+H)⁺, 1019.6 (2M+H)⁺, 492.6 (M−OH⁻)⁺

EXAMPLE 53

To a mixture of compound of formula (45) (1.20 g, 1.0 eq), preparedaccording to example 52, compound of formula (CG1MR-IV-1) (0.69 g, 1.1eq), prepared according to example 8, and DMF (25 ml) at RT, DIPEA (0.34g, 1.1 eq) was added. The resulting mixture was stirred at RT for 2hours. Then the DMF was removed under vacuum to afford a slightly yellowresidue which was then mixed with acetone (50 ml) and stirred at RT for1 hour. The mixture was filtered, the cake was washed with acetone (2times with 10 ml each), then dried under vacuum to afford 1.12 g ofcompound of formula (35) (72% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 1.20 (3H, d, J=7.2 Hz), 1.24 to1.40 (4H, m), 1.58 to 1.75 (2H, m), 1.78 (3H, s), 1.87 (3H, s), 2.33(2H, t, J=7.2 Hz), 2.98 to 3.03 (2H, m), 3.16 to 3.21 (2H, m), 3.46 (2H,t, J=5.8 Hz), 3.60 (2H, t, J=7.2 Hz), 3.72 to 3.75 (2H, m), 4.04 (2H, t,J=4.4 Hz), 4.24 to 4.36 (2H, m), 4.44 (2H, d, J=5.6 Hz), 4.86 (1H, t,J=5.6 Hz), 7.00 (2H, s), 7.16 to 7.18 (11-1, m), 7.26 to 7.28 (1H, m),7.34 (1H, d, J=1.4 Hz), 7.80 (1H, t, J=5.4 Hz), 7.99 (1H, d, J=8.0 Hz),8.03 (1H, t, J=5.4 Hz), 8.10 (1H, d, J=6.8 Hz), 9.79 (1H, s).

ESI-MS: 661.5 (M+H)⁺, 643.6 (M−OH⁻)⁺

EXAMPLE 54

A mixture of compound of formula (35) (400.0 mg, 1.0 eq), preparedaccording to example 53, 4 angstrom molecular sieves (800.0 mg),anhydrous DMF (4.0 ml) and compound of formula (II-1) (373.0 mg, 2.0 eq)was stirred at RT for 5 min. Then DIPEA (313.5 mg, 4.0 eq) was added.The resulting mixture was stirred at RT for 5 hours. Then compound offormula (DOXO) as HCl salt (328.0 mg, 0.9 eq) was added and the mixturewas then stirred for 4 hours. Then MeCN (20 ml) was added. A precipitatehad formed and was filtered and washed with a mixture of MeCN and DMF(5:1 (v/v), 2 times with 10 ml each). The filtrate was combined anddried under vacuum at 45° C. to get a hard residue. The residue wasdissolved in the mixture of DCM and MeOH (7:1, v/v, 5 ml) and purifiedby preparative silica gel TLC (DCM:MeOH=7:1, (v/v), Rf=0.15). Theproduct was extracted from the silica gel by a mixture of acetone andwater (20:1, v/v, 10 times with 20 ml each). The combined extractionsolutions were dried under vacuum to afford the crude product as a redsolid. The cured product was then mixed with acetonitrile (10 ml), themixture was stirred at RT for 0.5 hour and then filtered. The cake waswashed with acetonitrile (5 ml) then dried under vacuum at RT to afford100.7 mg of compound of formula (25) as a red solid (13% yield).

¹H NMR (400 MHz, DMSO-d, 20° C.) δ 1.13 (3H, d, J=6.4 Hz), 1.20 (3H, d,J=7.2 Hz), 1.24 to 1.38 (4H, m), 1.48 to 1.51 (1H, m), 1.59 to 1.73 (2H,m), 1.77 (3H, s), 1.82 to 1.86 (4H, m), 2.12 to 2.23 (2H, m), 2.29 (2H,t, J=7.2 Hz), 2.89 to 3.02 (4H, m), 3.11 to 3.15 (2H, m), 3.41 (2H, t,J=5.6 Hz), 3.46 (1H, brs), 3.54 (2H, t, J=7.2 Hz), 3.68 to 3.76 (3H, m),3.98 (3H, s), 4.03 (2H, brs), 4.14 to 4.18 (1H, m), 4.22 to 4.34 (2H,m), 4.58 (2H, d, J=5.2 Hz), 4.70 (1H, d, J=5.8 Hz), 4.85 to 4.92 (4H,m), 5.22 (1H, brs), 5.46 (1H, s), 6.81 (1H, d, J=8.0 Hz), 6.95 (2H, s),7.14 to 7.20 (2H, m), 7.39 (1H, s), 7.61 to 7.64 (1H, m), 7.82 (1H, t,J=5.2 Hz), 7.88 to 7.91 (2H, m), 7.98 to 8.03 (2H, m), 8.12 (1H, d,J=6.8 Hz), 9.89 (1H, s), 13.24 (1H, s), 14.00 (1H, s).

ESI-MS: 1252.1 (M+Na)⁺

EXAMPLE 55

Compound of formula (6-1) as HCl salt (22.81 g, 1.0 eq.), preparedaccording to example 6, 2-[2-(2-Methoxyethoxy)ethoxy]acetic acid (8.00g, 0.9 eq.), purchased from Aldrich, TBTU (24.00 g, 1.5 eq), and DIPEA(16.00 g,) were dissolved in DMF (100 ml). The resulting solution wasstirred at RT for 20 h. The reaction mixture was concentrated todryness, the residue was then purified by silica gel chromatography(eluent DCM:MeOH=10:1 to 7:1 (v/v)) to afford 12.02 g of compound offormula (6c) as a slightly yellow solid (41% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.86 (6H, dd, J₁=24.4 Hz, J₂=6.8Hz), 1.37 (9H, s), 1.39 to 1.52 (2H, m), 1.56 to 1.75 (4H, m), 1.97 to2.06 (1H, m), 2.91 to 3.07 (4H, m), 3.19 to 3.22 (2H, m), 3.42 to 3.47(2H, m), 3.52 to 3.64 (6H, m), 3.81 (3H, s), 3.95 (2H, brs), 4.30 to4.34 (1H, m), 4.36 to 4.41 (1H, m), 4.53 (2H, s), 5.43 (2H, s), 6.01(1H, t, J=5.8 Hz), 6.78 (1H, t, J=5.2 Hz), 7.34 to 7.40 (2H, m), 7.45(1H, d, J=8.8 Hz), 7.79 (1H, d, J=8.4 Hz), 8.04 (1H, t, J=5.6 Hz), 8.38(1H, d, J=7.2 Hz), 10.36 (1H, s).

ESI-MS: 584.7 (M+H)⁺, 1167.2 (2M+H)⁺

EXAMPLE 56

A mixture of compound of formula (6c) (8.01 g, 1.0 eq.), preparedaccording to example 55, compound of formula (SGM-III-2) (6.89 g, 2.0eq.), prepared according to example 24, K₂CO₃ (3.80 g, 2.0 eq.), andanhydrous DMF (60 ml) was heated to 50° C. and stirred under nitrogenatmosphere for 7 days. Then the reaction mixture was concentrated todryness under vacuum. Dichloromethane (15 ml) was added to the residue,the resulting mixture was purified by silica gel column chromatography(eluent DCM:MeOH=7:1 (v/v)) to afford 5.52 g of compound of formula(56-1) as a slightly yellow solid (50% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) δ 0.86 (6H, dd, J₁=24.4 Hz, J₂=6.8Hz), 1.37 (9H, s), 1.39 to 1.52 (2H, m), 1.56 to 1.75 (4H, m), 1.97 to2.06 (1H, m), 2.91 to 3.07 (4H, m), 3.19 to 3.22 (2H, m), 3.42 to 3.47(2H, m), 3.52 to 3.64 (6H, m), 3.81 (3H, s), 3.95 (2H, brs), 4.30 to4.34 (11H, m), 4.36 to 4.41 (1H, m), 4.53 (2H, s), 5.43 (2H, s), 6.01(1H, t, J=5.8 Hz), 6.78 (1H, t, J=5.2 Hz), 7.34 to 7.40 (2H, m), 7.45(1, d, J=8.8 Hz), 7.79 (1H, d, J=8.4 Hz), 8.04 (1H, t, J=5.6 Hz), 8.38(1H, d, J=7.2 Hz), 10.36 (1H, s).

ESI-MS: 698.7 (M−^(t)BuOCO+2H)⁺, 798.3 (M+H)⁺, 820.7 (M+Na)⁺, 1595.7(2M+H)⁺

EXAMPLE 57

Compound of formula of (56-1), prepared according to example 56, wassuspended in 10% (w/w) HCl in 1,4-dioxane (60 ml). The resulting mixturewas stirred at RT for 8 hours then concentrated to afford 4.99 g ofcompound of formula (56) as a white solid (quantitative yield).

ESI-MS: 698.4 (M+H)⁺, 1394.6 (2M+H)⁺

EXAMPLE 58

A mixture of compound of formula (56) as HCl salt (4.91 g, 1.0 eq.),prepared according to example 57, water (45 ml), CaCl₂ (1.49 g, 2.0 eq.)and NaBH₄ (1.02 g, 4.0 eq.) was stirred at RT. Further amount of NaBH₄was added portionwise (1.02 g, 4.0 eq. after a total of 2.5 hours ofstirring; 1.01 g, 4.0 eq. after a total of 4 hours of stirring; 1.03 g,4.0 eq. after a total of 6 hours of stirring). After a total of 22 hoursof stirring, MeOH (20 ml) was added to the mixture. The reaction mixturewas then filtered and the wet cake was washed with MeOH (3 times with 15ml each). The filtrate was collected and combined and then evaporated todryness. The residue was purified by silica gel column chromatography(eluent DCM:MeOH:Et₃N=80:20:2.5 (v/v)) to afford 1.12 g of compound offormula (46) as a slightly yellow solid (25% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) δ 0.86 (6H, dd, J₁=22.8 Hz, J₂=6.8Hz), 1.32 to 1.48 (4H, m), 1.51 to 1.73 (4H, m), 1.96 to 2.06 (1H, m),2.58 (2H, t, J=6.8 Hz), 2.91 to 3.06 (2H, m), 3.16 to 3.21 (2H, m), 3.22(3H, s), 3.52 to 3.64 (8H, m), 3.95 (2H, brs), 4.29 to 4.33 (1H, m),4.36 to 4.41 (2H, m), 4.46 (2H, s), 4.51 (2H, s), 5.44 (2H, s), 6.10(1H, t, J=5.6 Hz), 7.22 to 7.27 (3H, m), 7.48 (1H, d, J=8.8 Hz), 8.19(1H, t, J=5.4 Hz), 8.34 (1H, d, J=7.6 Hz), 10.05 (1H, s).

ESI-MS: 652.4 (M−OH⁻)⁺, 670.4 (M+H)⁺, 1338.7 (2M+H)⁺

EXAMPLE 59

To a mixture of compound of formula (46) (250.7 mg, 1.0 eq), preparedaccording to example 58, compound of formula (CG1MR-IV-1) (103.1 g, 1.1eq), prepared according to example 8, and DMF (4 ml) at RT, DIPEA (53.6mg, 1.1 eq) was added. The resulting mixture was stirred at RT for 1hour. Then the DMF was removed under vacuum to afford a slightly yellowresidue which was then mixed with acetone (5 ml) and stirred at RT for 1hour. The mixture was filtered, the cake was washed with acetone (3times with 3 ml each), then dried under vacuum to afford 253.7 g ofcompound of formula (36) (83% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.85 (6H, dd, J₁=23.0 Hz, J₂=6.8Hz), 1.36 to 1.54 (41-1, m), 1.58 to 1.72 (2H, m), 1.97 to 2.05 (1H, m),2.32 (2H, t, J=7.2 Hz), 2.92 to 3.05 (4H, m), 3.08 to 3.13 (2H, m), 3.22(3H, s), 3.44 (2H, t, J=4.8 Hz), 3.52 to 3.62 (8H, m), 3.95 (2H, s),4.29 to 4.33 (1H, m), 4.35 to 4.40 (1H, m), 4.46 (2H, s), 4.52 (2H, d,J=5.2 Hz), 5.08 (1H, t, J=5.6 Hz), 5.42 (2H, s), 6.01 (1H, t, J=5.2 Hz),6.99 (2H, s), 7.20 to 7.27 (3H, m), 7.46 (1H, d, J=8.8 Hz), 7.95 (1H, t,J=5.4 Hz), 8.05 (1H, t, J=5.8 Hz), 8.31 (1H, d, J=7.6 Hz), 10.00 (1H,s).

ESI-MS: 803.4 (M−OH⁻)⁺, 821.1 (M+H)⁺, 1641.1 (2M+H)⁺

EXAMPLE 60

A mixture of compound of formula (36) (201.3 mg, 1.0 eq), preparedaccording to example 59, 4 angstrom molecular sieves (400.0 mg),anhydrous DMF (4.0 ml) and compound of formula (II-1) (146.9 mg, 2.0 eq)was stirred at RT for 10 min. Then DIPEA (105.7 mg, 3.3 eq) was added.The resulting mixture was stirred at RT for 4.5 hours. Then compound offormula (DOXO) as HCl salt (142.1 mg, 1.0 eq) was added and the mixturewas then stirred for 2.5 hours. Then MeCN (20 ml) was added. Aprecipitate had formed and was filtered and washed with a mixture ofMeCN and DMF (5:1 (v/v), 4 times with 4 ml each). The filtrate wascombined and dried under vacuum at 45° C. to get a hard residue. Theresidue was dissolved in the mixture of DCM and MeOH (7:1, v/v, 3 ml)and purified by preparative silica gel TLC (DCM:MeOH=7:1, (v/v),Rf=0.3). The product was extracted from the silica gel by a mixture ofacetone and water (10:1, v/v, 6 times with 10 ml each). The combinedextraction solutions were dried under vacuum to afford the crude productas a red solid. The cured product was then mixed with acetonitrile (5ml), the mixture was stirred at RT for 2 hour and then filtered. Thecake was washed with acetonitrile (2 times with 2 ml each) then driedunder vacuum at RT to afford 33.2 mg of compound of formula (26) as ared solid (10% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.84 (6H, dd, J₁=23.2 Hz, J₂=6.8Hz), 1.13 (3H, d, J=6.4 Hz), 1.30 to 1.70 (7H, m), 1.82 to 1.87 (4H, m),1.97 to 2.04 (1H, m), 2.08 to 2.22 (2H, m), 2.28 (2H, t, J=7.2 Hz), 2.91to 3.09 (8H, m), 3.22 (3H, s), 3.42 to 3.46 (3H, m), 3.52 to 3.61 (8H,m), 3.70 to 3.76 (1H, m), 3.95 (2H, s), 3.99 (3H, s), 4.13 to 4.18 (2H,m), 4.28 to 4.32 (1H, m), 4.34 to 4.41 (3H, m), 4.58 (2H, d, J=5.6 Hz),4.70 (1H, d, J=5.2 Hz), 4.85 (1H, t, J=5.8 Hz), 4.94 (1H, brs), 5.03(2H, s), 5.22 (1H, brs), 5.41 (2H, s), 5.46 (1H, brs), 5.99 (1H, t,J=5.4 Hz), 6.86 (1H, d, J=8.0 Hz), 6.97 (2H, s), 7.19-7.24 (3H, m), 7.44(1H, d, J=8.8 Hz), 7.65 (1H, t, J=4.8 Hz), 7.85 to 7.91 (4H, m), 8.30(1H, d, J=7.2 Hz), 10.04 (1H, s), 13.26 (1H, s), 14.02 (1H, s).

ESI-MS: 1390.2 (M+H)⁺

EXAMPLE 61

A mixture of compound of formula (TAXO) (200.0 mg, 1.0 eq), 4 angstrommolecular sieves (100.0 mg), anhydrous DCM (4.0 ml) and compound offormula (II-1) (146.9 mg, 2.0 eq) at 0° C. was stirred for 10 min. Tothe mixture was then added pyridine (28.1 mg, 1.5 eq). The resultingmixture was further stirred for 41 hours at 0° C. under N₂. To themixture was then added N,N′-Dimethyl-1,2-ethylenediamine (102.0 mg, 5.0eq.) at 0° C. under N₂. After stirred for 2 hours, the mixture wasfiltered. The cake was washed with dichloromethane (1 mL). The filtratewas combined then evaporated to dryness to give yellow residue. Theresidue was further purified by preparative silica gel TLC(DCM:MeOH=7:1, (v/v)) to afford 220.0 mg of compound of formula(TAXO-t1-1) as a slightly yellow solid in 96% isolated yield.

ESI-MS: 968.5 (M+Na)⁺

EXAMPLE 62

A mixture of compound of formula (31) (252.0 mg, 1.2 eq), preparedaccording to example 21, compound of formula (II-1) (136.0 mg, 1.8 eq),4 angstrom molecular sieves (100.0 mg) and anhydrous DMF (1 ml) wasstirred at RT for 10 min. Then DIPEA (80.1 mg, 2.5 eq) was added. Theresulting mixture was stirred for 2 hours at RT. Then compound offormula (XXXX), prepared according to example 61, was added. Theresulting mixture was stirred for 2 hours. The resulting suspension wasfiltered; the cake was further washed with DCM (3 times with 10 mleach). The filtrate was combined then evaporated to dryness at 35° C.The resulting residue was mixed with DCM (30 ml) and stirred for 0.5hour. The resulting suspension was filtered, the cake was further washedwith DCM (2 times with 5 ml each). The filtrate was dried under vacuumat RT. The crude product was dissolved in the mixed solvent ofCH₂Cl₂-MeOH (8:1, (v/v)) and further purified by preparative silica gelTLC (DCM:MeOH=8:1, (v/v)). The product was extracted from the silica-gelby a mixture of THF-water (20:1 (v/v), 6 times with 30 ml each), driedunder vacuum to afford the crude product as a white solid. The crudeproduct was then mixed with Et₂O (5 ml), stirred at 25° C. for 0.5 hourthen filtered. The cake was washed with Et₂O (3 times with 5 ml each)then dried under vacuum at RT to afford 100.0 mg of compound of formula(21-TAXO-t1-1) as a white solid in 21% isolated yield.

ESI-MS: 1842.1 (M+H)⁺, 1864.5 (M+Na)⁺

COMPARATIVE EXAMPLE 1

T a mixture of Boc-Cit-OH (1.00 g, 1.0 eq.), prepared according toexample 2, EEDQ (1.35 g, 1.5 eq.) and THF (15 ml), p-amino benzoic acidmethyl ester (0.82 g, 1.5 eq.) was added. The resulting mixture wasstirred at RT for 14 hours. Then the solvent was removed under vacuumand the residue was purified by silica gel chromatography (eluentPE:EtOAc=6:1 (v/v) then DCM:MeOH=10:1 (v/v)) to afford 1.2 g of compoundof formula (Comp-6-4) as a white solid (81% yield).

Analysis by silica gel TLC:eluent EtOAc (Rf=0.35, UV254)

¹H NMR (400 MHz, CDCl₃, 20° C.) delta 1.28 to 1.45 (11H, m), 1.55 to1.65 (2H, m), 2.91 to 3.08 (2H, m), 3.83 (3H, s), 4.09 to 4.14 (1H, m),5.43 (2H, s), 5.99 (1H, t, J=5.6 Hz), 7.10 (1H, d, J=7.6 Hz), 7.75 to7.77 (2H, m), 7.91 to 7.93 (2H, m), 10.32 (1H, s).

ESI-MS: 309.3 (M−tBuOCO+2H)+, 409.2 (M+H)+, 817.1 (2M+H)+

COMPARATIVE EXAMPLE 2

A mixture of compound of formula (Comp-6-4) (1.66 g), prepared accordingto comparative example 1, and of a solution of 15% (w/w) HCl in1,4-dioxane (10 ml) was stirred at RT for 3 hours. The reaction mixturewas then concentrated under vacuum to afford 1.38 g of compound offormula (Comp-6-3) as HCl salt, being a white solid (98% yield).

COMPARATIVE EXAMPLE 3

To a mixture of compound of formula (Comp-6-3) as HCl salt (14.50 g, 1.0eq.), prepared according to comparative example 2, Boc-L-Val (10.05 g,1.1 eq.), TBTU (27.00 g, 2.0 eq.) and DMF (80 ml) was added DIPEA (16.31g, 3.0 eq.). The resulting mixture was stirred at RT for 16 hours. Thenthe mixture was diluted with water (120 ml), and then extracted withEtOAc (5 times with 100 ml each), the organic phases were combined andevaporated to dryness, the resulting residue was purified by silica gelcolumn chromatography (eluent DCM:MeOH=20:1 to 10:1 to 7:1 (v/v)) toafford 12.9 g of compound of formula (Comp-6-2) as a solid (60% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.85 (6H, dd, J1=17.4 Hz, J2=6.6Hz), 1.39 (9H, s), 1.43 to 1.50 (2H, m), 1.57 to 1.76 (2H, m), 1.91 to1.97 (1H, m), 2.91 to 3.09 (2H, m), 3.83 (3H, s), 3.85 to 3.88 (1H, m),4.43 to 4.48 (1H, m), 5.43 (2H, s), 5.99 (1H, t, J=5.8 Hz), 6.72 (1H, d,J=8.8 Hz), 7.73 to 7.76 (2H, m), 7.90 to 7.94 (2H, m), 8.07 (1H, d,J=7.6 Hz), 10.39 (1H, s).

COMPARATIVE EXAMPLE 4

A mixture of compound of formula (Comp-6-2) (12.3 g), prepared accordingto comparative example 3, and of a solution of 10% (w/w) HCl in dioxane(60 ml) was stirred at RT for 3 hours. The mixture was then concentratedunder vacuum to afford 10.9 g of compound of formula (Comp-6-1) as HClsalt, being a white solid (quantitative yield).

COMPARATIVE EXAMPLE 5

To a mixture of compound of formula (Comp-6-1) as HCl salt (10.20 g, 1.0eq.), prepared according to comparative example 4, and anhydrous THF(150 ml) at −20° C. was added a solution of DIBAL-H in hexanes (1 M,120.0 ml, 6.2 eq.). The mixture was warmed to RT and stirred for 14hours. Then MeOH (20 ml) was added, followed by a saturated aqueouspotassium sodium tartrate solution (180 ml) was added and the mixturewas stirred for 30 min at RT. The resulting mixture was evaporated todryness to afford a white residue which was washed with MeOH (5 timeswith 50 ml each). The eluents were combined, concentrated and purifiedby silica gel column chromatography (eluent DCM:MeOH=10:1 to 7:1 to 5:1(v/v)) to afford 5.10 g or compound of formula (Comp-10) as a whitesolid (58% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.89 (6H, dd, J1=24.0 Hz, J2=6.8Hz), 1.33 to 1.50 (2H, m), 1.56 to 1.77 (2H, m), 1.96 to 2.04 (1H, m),2.92 to 3.07 (2H, m), 3.30 (1H, d, J=5.2 Hz), 4.12 (2H, brs), 4.43 (2H,s), 4.47 to 4.52 (1H, m), 5.12 (1H, brs), 5.50 (2H, s), 6.10 (1H, t,J=5.6 Hz), 7.24 (2H, d, J=8.8 Hz), 7.54 to 7.57 (2H, m), 8.37 (1H, d,J=5.8 Hz), 10.11 (1H, s).

ESI-MS: 380.4 (M+H)+, 759.2 (2M+H)+

COMPARATIVE EXAMPLE 6

To a mixture of compound of formula (Comp-10) (1.02 g, 1.0 eq.),prepared according to comparative example 5, compound of formula(CG1MR-IV-2) (0.92 g, 1.1 eq.)

and DMF (15 ml) at RT was added DIPEA (0.39 g, 1.1 eq.). The resultingmixture was stirred at RT for 16 hours. Then the DMF was removed undervacuum to afford a slightly yellow residue. The residue was then mixedwith acetone (20 ml) and the mixture was then stirred at RT for 5 hours.The mixture was filtered, the wet cake was washed with acetone (2 timeswith 10 ml each) and then dried under vacuum to afford 1.05 g ofcompound of formula (Comp-11) as a slightly yellow solid (67% yield).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.84 (6H, dd, J1=12.2 Hz, J2=6.6Hz), 1.17 to 1.31 (2H, m), 1.37 to 1.75 (8H, m), 1.95 to 2.00 (1H, m),2.11 to 2.21 (2H, m), 2.92 to 3.06 (2H, m), 3.38 (2H, t, J=6.8 Hz), 4.18to 4.22 (1H, m), 4.38 to 4.39 (1H, m), 4.43 (2H, d, J=4.8 Hz), 5.09 (1H,t, J=5.2 Hz), 5.41 (2H, s), 5.98 (1H, t, J=5.6 Hz), 7.00 (2H, s), 7.23(2H, d, J=8.0 Hz), 7.55 (2H, d, J=8.2 Hz), 7.81 (2H, d, J=8.4 Hz), 8.06(1H, d, J=7.2 Hz), 9.90 (1H, s).

COMPARATIVE EXAMPLE 7

A mixture of compound of formula (Comp-11) (350.0 mg, 1.0 eq.), preparedaccording to comparative example 6, DIPEA (307.0 mg, 3.9 eq.), 500.0 mg4 angstrom molecular sieves and anhydrous DMF (5.0 ml) was stirred for25 min. Then compound of formula (II-1) (280.0 mg, 1.5 eq.) was added.The resulting mixture was stirred at RT for 3 hours. Then compound offormula (DOXO) as HCl salt (368.8 mg, 1.0 eq.) was added and then themixture was stirred for 12 hours. Then MeCN (25.0 ml) was added to thereaction mixture. A precipitate had formed and was filtered and washedwith a mixture of MeCN and DMF (5:1 (v/v), 3 times with 5 ml each). Thefiltrates were combined and dried under vacuum at 45° C. to afford adark red residue. The residue was dissolved in a mixture of DCM and MeOH(7:1 (v/v)) and purified by preparative silica gel TLC (DCM:MeOH=7:1(v/v), Rf=0.15). The product was extracted from the silica gel by amixture of acetone and water (20:1 (v/v), 10 times with 50 ml each), thecombined extracts were dried under vacuum to afford the crude product asa red solid. The crude product was then mixed with acetonitrile (20 ml),the mixture was stirred at RT for 18 hours then filtered. The cake wasmixed with acetonitrile (10 ml) and the mixture was stirred for 3 hoursat RT. The mixture was then filtered. The cake was dried under vacuum atRT to afford 61.0 mg of compound of formula (Comp-12) as a red solid(8.7% yield);

in formula (Comp-12) doxorubicin is the compound of formula (DOXO),which is connected via the amino group denoted with (d1) in formula(Comp-12) and in formula (DOXO).

¹H NMR (400 MHz, DMSO-d₆, 20° C.) delta 0.85 (6H, dd, J1=12.0 Hz, J2=6.8Hz), 1.12 (3H, d, J=6.4 Hz), 1.16 to 1.22 (2H, m), 1.31 to 1.50 (7H, m),1.54 to 1.71 (2H, m), 1.82 to 1.88 (1-, m), 1.91 to 2.01 (1H, m), 2.10to 2.22 (4H, m), 2.89 to 3.03 (4H, m), 3.38 (2H, t, J=6.8 Hz), 3.44 (1H,m), 3.71 to 3.75 (1H, m), 3.99 (3H, s), 4.16 to 4.20 (2H, m), 4.33 to4.39 (1H, m), 4.58 (2H, d, J=5.6 Hz), 4.71 (1H, d, J=5.6 Hz), 4.86 (1H,t, J=6.0 Hz), 4.89 (2H, s), 4.95 (1H, brs), 5.22 (1H, d, J=2.8 Hz), 5.40(2H, s), 5.47 (1H, s), 5.99 (1H, t, J=4.8 Hz), 6.84 (1H, d, J=8.0 Hz),7.00 (2H, s), 7.24 (2H, d, J=8.4 Hz), 7.55 (2H, d, J=8.4 Hz), 7.66 (1H,t, J=4.8 Hz), 7.80 (1H, d, J=8.4 Hz), 7.92 (2H, d, J=4.8 Hz), 8.06 (1H,d, J=7.6 Hz), 9.97 (1H, s), 13.28 (1H, s), 14.04 (1H, s).

ESI-MS: 1141.7 (M+H)+

EXAMPLE 100

General Method Description for the Preparation of Compounds of Formula(I) Given in Table 1:

To a 10 mM aqueous solution of N-acetyl-cysteine (2500 μl, 5 eq.) wasadded a 10 mM solution of the respective compound of formula (II) inN,N-dimethylacetamide (500 μl). The pH was adjusted to 7.5 with a 0.3 Msodium hydrogen phosphate solution, and the reaction mixture stirred for2 h at 20° C. The resulting respective solution of compound of formula(I) was used/tested without further purification.

Details are given in Table (1)

TABLE 1 Example Compound of formula (II) Compound of formula (I) 100-1Compound of formula (Comp-12), Compound of formula prepared according tocomparative (Comp-13) example 7 100-2 Compound of formula (23), Compoundof formula (13) prepared according to example 36 100-3 Compound offormula (20), Compound of formula (10) prepared according to example 15100-4 Compound of formula (21), Compound of formula (11) preparedaccording to example 22 100-5 Compound of formula (22), Compound offormula (12) prepared according to example 29 100-6 Compound of formula(25), Compound of formula (15) prepared according to example 54 100-7Compound of formula (26), Compound of formula (16) prepared according toexample 60 100-8 Compound of formula (24), Compound of formula (14)prepared according to example 44

Method RP-HPLC

RP-HPLC analysis of the respective solution of compounds of formula (I),prepared according to example 100, was done with the followingparameter:

Luna 5 u C18 250×4.6 mm column (purchased form Phenomenex), solvent A:0.1% (v/v) TFA in water, solvent B: 0.1% (v/v) TFA in acetonitrile, 100%solvent A for 10 min., gradient from 0 to 70% solvent B over next 70min., and to 100% over next 3 min., 1 ml/min., detection at 254 nm.

RT=retention time

Values are given in Table 2

TABLE 2 RT compound of formula (DOXO) 44.9 min. compound of formula(Comp-13) 54.5 min. compound of formula (13) 55.1 min. compound offormula (10) 51.2 min. compound of formula (11) 51.7 min. compound offormula (12) 50.7 min. compound of formula (15) 50.6 min. compound offormula (16) 52.2 min. compound of formula (14) 48.3 min.Solubility Test

The respective solution of compound of formula (I) (15, 48 and 96 μlrespectively), prepared according to example 100 were mixed with water(135, 102 and 54 μl respectively) to get a total of 150 μl of therespective three diluted solutions. These three diluted solutions have aconcentration of 1.5%, 5.0% and 10.0% of DMA respectively, the % being %by volume, based on the total volume of the water in the respectivesolutions. These diluted solutions were stirred for 1 h at 20° C. andthen analysed by Method RP-HPLC.

The solubility of the compounds of formula (I) was assessed bycomparison of the peak areas (relative to the most soluble conjugate,namely compound of formula (11), the peak area of which was set to 100%,denoted with (*ref) in Table 3), values are given in Table 3:

TABLE 3 1.5% 5.0% 10.0% DMA DMA DMA Compound of formula (Comp-13)  8%41% 77% Compound of formula (13) 17% 53% 82% Compound of formula (10)15% 47% 84% Compound of formula (11) 19% 58% 100% (*ref) Compound offormula (12) 16% 49% 85% Compound of formula (15) 16% 45% 77% Compoundof formula (16) 16% 47% 80% Compound of formula (14) 12% 36% 66%

The higher solubilities, especially at the more relevant low DMAconcentration, of the compound of formula (I) compared to the referencelinker compound of formula (Comp-13) provide two advantages:

-   -   1) A higher yield when compound of formula (I) is synthesized,        thanks to reduced aggregation    -   2) Superior pharmacokinetic        Cathepsin B Release of Compound of Formula (DOXO)

Bovine spleen cathepsin B (SAFC C6286-10UN, 10 units) was dissolved in 1ml of a pH 5.0 aqueous acetate buffer (25 mM acetate and 1 mM EDTA) toprovide for a cathepsin B stock solution.

This cathepsin B stock solution (16 μl) was mixed with a aqueoussolution of 30 mM dithiothreitol and 15 mM EDTA (32 μl) and theresulting solution was left to stand without stirring for 15 min. at 20°C. Then an aqueous solution of 25 mM acetate and 1 mM EDTA (1175 μl),the respective solution of compound of formula (I) (142 μl, preparedaccording to example 100), N,N-dimethylacetamide (53.7 μl), and a 10 mMaqueous solution of daunorubicin as internal standard for assigningpeaks based on retention time (157.7 μl) were added. The resultingsolution was incubated for 2 days at 37° C. Aliquots (100 μl) wereperiodically removed as given in Table 4 and analysed undiluted byMethod RP-HPLC. The relative percentage of released compound of formula(DOXO) relative to respective compound of formula (I) during theexperiment are given in Table 4:

TABLE 4 0 h 4 h 8 h 24 h 48 h Compound of <0.1% 18.0% 24.4% 32.2% 33.8%formula (13) Compound of <0.1% 15.8% 23.5% 35.5% 41.4% formula (10)Compound of <0.1% 6.4% 8.7% 13.5% 15.4% formula (11) Compound of <0.1%3.0% 3.8% 5.3% 5.0% formula (12) Compound of <0.1% 1.3% 1.6% 2.3% 4.1%formula (15) Compound of <0.1% 0.6% 1.0% 2.0% 2.2% formula (16) Compoundof <0.1% <0.1% 1.0% 1.7% 2.2% formula (14)

All compounds showed drug release in the presence of Cathepsin B.

Stability in Human Serum

To human serum (SAFC H4522, 950 μl) was added the respective solution ofcompound of formula (I) (950 μl, prepared according to example 100) anda 10 mM aqueous solution of daunorubicin as internal standard (100 μl).The resulting solution was incubated at 37° C. for 7 days. Aliquots (100μl) were periodically removed as given in Table 4, diluted with 0 to 5°C. methanol (400 μl), filtered, and the resulting filtrate analyzed byMethod RP-HPLC. Released compound of formula (DOXO) was quantifiedrelative to the daunorubicin (the internal standard), values are givenin Table 5:

TABLE 5 0 h 4 h 8 h 24 h 48 h 72 h Compound of formula (13) <0.1% <0.1%<0.1% 4.4% 6.3% 6.9% Compound of formula (10) <0.1% <0.1% <0.1% 2.4%4.3% 4.3% Compound of formula (11) <0.1% <0.1% <0.1% <0.1% <0.1% <0.1%Compound of formula (12) <0.1% <0.1% <0.1% <0.1% <0.1% <0.1% Compound offormula (15) <0.1% <0.1% 0.2% 3.2% 5.2% 6.0% Compound of formula (16)<0.1% 1.3% 1.2% 1.9% 3.2% 1.6% Compound of formula (14) <0.1% <0.1%<0.1% 1.1% 2.5% 2.6%

All branched linkers proved to have good stability.

EXAMPLE 101

A compound of formula (12-101), derived from compound of formula (22)and monoclonal anti-interleukin-1b antibody was prepared as follows:

Monoclonal anti-interleukin-1b antibody produced in mouse (5 mg, Sigmaarticle No 13642) was mixed in PBS (8.0 ml).

To an aliquot of this solution (3 ml) was added a 1.0 mM aqueoussolution of tris(2-carboxyethyl)phosphine hydrochloride (25 μl, 2.0 eq.)and the mixture was stirred for 90 min. at 20° C. A 1.0 mM solution ofcompound of formula (22), prepared according to example 29, inN,N-dimethylacetamide (64.4 μl, 5.15 eq.) was added and the resultingmixture further stirred for 30 min at 20° C. A 1.0 mM aqueous solutionof N-acetyl-cysteine (64.4 μl, 5.15 eq.) was added and the resultingmixture further stirred for 36 min. at 20° C. to yield a so calledconjugation mix. A NAP-25 column was rinsed with PBS (25 ml), loadedwith the conjugation mix (2.5 ml) and eluted with PBS (5.0 ml).Fractions were collected and those that contained protein were pooled.The pooled protein solution, which comprised compound of formula(12-101), was analysed by Method SEC-HPLC (results are given in Table 6)and by Method HIC with the monoclonal anti-interleukin-1b antibodysuspended in PBS.

EXAMPLE 102

A compound of formula (15-102), derived from compound of formula (25)and monoclonal anti-interleukin-1b antibody was prepared as follows:

Monoclonal anti-interleukin-1b antibody produced in mouse (5 mg, Sigmaarticle No 13642, as used in example 101) was mixed in PBS (8.0 ml). Toan aliquot of this solution (3 ml) was added a 1.0 mM aqueous solutionof tris(2-carboxyethyl)phosphine hydrochloride (25 μl, 2.0 eq.) and themixture was stirred for 90 min. at 20° C. A 1.0 mM solution of compoundof formula (25), prepared according to example 54, inN,N-dimethylacetamide (64.4 μl, 5.15 eq.) was added and the mixturefurther stirred for 30 min. at 20° C. A 1.0 mM aqueous solution ofN-acetyl-cysteine (64.4 μl, 5.15 eq.) was added and the mixture furtherstirred for 42 min. at 20° C. to yield a so called conjugation mix. ANAP-25 column was rinsed with PBS (25 ml), loaded with the conjugationmix (2.5 ml) and eluted with PBS (5.0 ml). Fractions were collected andthose that contained protein were pooled. The pooled protein solution,which comprised compound of formula (15-102), was analysed by MethodSEC-HPLC (results are given in Table 6) and by Method HIC with themonoclonal anti-interleukin-1b antibody suspended in PBS.

Method SEC-HPLC

SEC-HPLC analysis of respective pooled protein solution was done withthe following parameters:

TSK G3000SWXL 300×7.8 mm column (Silica based column with mean pore sizeof 250 Angstrom and mean particle size of 5 micrometer, purchased fromTosoh Bioscience), eluent 10% (v/v) isopropanol in 0.2 M potassiumphosphate buffer, 0.5 ml/min., detection at 280 nm. RT=retention time

The peak and therewith the retention time of the respective compound offormula (I) was identified by comparison with the unmodified Monoclonalanti-interleukin-1b antibody, which was used as substrate in theexamples 101 and 102.

HMW=high molecular weight fraction, not assigned

LMW=low molecular weight fraction, not assigned

TABLE 6 RT purity HMW LMW Monoclonal anti-interleukin- 15.5 min. 92.1%7.6% 0.3% 1b antibody as used in examples 101 and 102 Compound offormula (12-101), 15.6 min. 92.4% 7.5% 0.1% prepared according toexample 101 Compound of formula (15-102), 15.6 min. 91.5% 8.4% 0.1%prepared according to example 102Method HIC

HIC analysis of respective pooled protein solution was done with thefollowing parameters: TSK-gel Butyl-NPR 4.6 mm×35 mm column (NPR meansnon-porous resin, polymethacrylate base material with mean particle size2.5 micrometer, purchased from Tosoh Bioscience), solvent A: 50 mMsodium phosphate buffer, solvent B: 25% (v/v) isopropanol in 50 mMsodium phosphate buffer, gradient from 100% solvent A to 100% solvent Bover 12 min., 0.8 ml/min, detection at 280 nm.

FIG. 1 shows the obtained chromatogram.

Legend of FIG. 1:

-   Continuous line    The monoclonal anti-interleukin-1b antibody used in examples 101 and    102-   Dashed line    Compound of formula (12-101), prepared according to example 101-   Dotted line    Compound of formula (15-102), prepared according to example-   AU absorption unit

The invention claimed is:
 1. A compound of formula (VI):

wherein AA¹ represents one alpha amino acid residue in which a carboxylterminal thereof forms the shown amide bond to NH;

the alpha amino acid residue, AA¹, having an alpha amino group denotedby structure (3); R1 and R2 are identical or different and independentlyfrom each other selected from the group consisting of hydrogen and PGN;PGN is a protecting group selected from the group consisting of Boc,Fmoc and benzyloxycarbonyl; and AA¹ is a residue of an amino aidselected from the group consisting of alanine, valine, leucine,isoleucine, methionine, phenylalanine, tryptophan, lysine, lysine withside chain amino group protected with acetyl or formyl groups, arginine,histidine, ornithine, ornithine with side chain amino group protectedwith acetyl or formyl groups, and citrulline.
 2. A compound selectedfrom the group consisting of the following compounds:

wherein R1 and R2 are identical or different and independently from eachother selected from the group consisting of hydrogen and PGN; and PGN isa protecting group selected from the group consisting of Boc, Fmoc andbenzyloxycarbonyl.
 3. A compound according to claim 2, wherein thecompound is selected from the group consisting of a compound of formula(6-3), a compound of formula (6-4), a compound of formula (6b-3), and acompound of formula (6b-4), as follows: